Valency platform molecules comprising carbamate linkages

ABSTRACT

This invention pertains generally to valency molecules, such as valency platform molecules which act as scaffolds to which one or more molecules may be covalently tethered to form a conjugate. More particularly, the present invention pertains to valency platform molecules which comprise a carbamate linkage (i.e., —O—C(═O)—N&lt;). In one aspect, the present invention pertains to valency platforms comprising carbamate linkages, which molecules have the structure of any one of Formulae I, II, or III, shown in FIG.  1.  In one aspect, the present invention pertains to valency platforms comprising carbamate linkages, which molecules have the structure of any one of Formulae IV, V, or VI, shown in FIG.  8.  The present invention also pertains to methods of preparing such valency platform molecules, conjugates comprising such valency platform molecules, and methods of preparing such conjugates.

RELATED APPLICATIONS

This application claims the priority benefit of provisional applicationU.S. Ser. No. 60/111,641, filed Dec. 9, 1998, the contents of which areincorporated herein in their entirety.

TECHNICAL FIELD

This invention pertains generally to valency molecules. The inventionalso relates to the field of valency platform molecules which act asscaffolds to which one or more molecules may be covalently tethered toform a conjugate. More particularly, the present invention pertains tovalency molecules which comprise a carbamate linkage (i.e.,—O—C(═O)—N<). In one aspect, the present invention pertains to valencyplatforms comprising carbamate linkages, which molecules have thestructure of any one of Formulae I, II, or III, shown in FIG. 1. In oneaspect, the present invention pertains to valency platforms comprisingcarbamate linkages, which molecules have the structure of any one ofFormulae IV, V, or VI, shown in FIG. 8. The present invention alsopertains to methods of preparing such valency molecules, conjugatescomprising such valency molecules, and methods of preparing suchconjugates.

BACKGROUND

A “valency platform” is a molecule with one or more (and typicallymultiple) attachment sites which can be used to covalently attachbiologically active molecules of interest to a common scaffold. Theattachment of biologically active molecules to a common scaffoldprovides multivalent conjugates in which multiple copies of thebiologically active molecule are covalently linked to the same platform.A “defined” or “chemically defined” valency platform is a platform withdefined structure, thus a defined number of attachment points and adefined valency. A defined valency platform conjugate is a conjugatewith defined structure and has a defined number of attached biologicallyactive compounds. Examples of biologically active molecules includeoligonucleotides, peptides, polypeptides, proteins, antibodies,saccharides, polysaccharides, epitopes, mimotopes, drugs, and the like.In general, biologically active compounds interact specifically withproteinaceous receptors.

Certain classes of chemically defined valency platforms, methods fortheir preparation, conjugates comprising them, and methods for thepreparation of such conjugates, have been described in the U.S. Pat.Nos. 5,162,515; 5,391,785; 5,276,013; 5,786,512; 5,726,329; 5,268,454;5,552,391; 5,606,047; and 5,663,395.

The valency platforms of the present invention reflect a new class ofvalency platforms which comprise a carbamate linkage, as shown, forexample, in Formulae I, II, and III in FIG. 1 and in Formulae IV, V, andVI in FIG. 8.

SUMMARY OF THE INVENTION

One aspect of the present invention pertains to a valency platformcompound having the structure of one of the following formulae:

wherein:

-   -   n is a positive integer from 1 to 10;    -   y¹, y², and y³ are independently 1 or 2;    -   J independently denotes either an oxygen atom or a covalent        bond;    -   R^(C) is selected from the group consisting of:        -   hydrocarbyl groups having from 1 to 20 carbon atoms;        -   organic groups consisting only of carbon, oxygen, and            hydrogen atoms, and having from 1 to 20 carbon atoms;        -   organic groups consisting only of carbon, oxygen, nitrogen,            and hydrogen atoms, and having from 1 to 20 carbon atoms;        -   organic groups consisting only of carbon, oxygen, sulfur,            and hydrogen atoms, and having from 1 to 20 carbon atoms;    -   each G¹, G², and G³ is independently selected from the group        consisting of:        -   hydrocarbyl groups having from 1 to 20 carbon atoms;        -   organic groups consisting only of carbon, oxygen, and            hydrogen atoms, and having from 1 to 20 carbon atoms;        -   organic groups consisting only of carbon, oxygen, nitrogen,            and hydrogen atoms, and having from 1 to 20 carbon atoms;    -   each R^(N) is independently selected from the group consisting        of:        -   hydrogen;        -   linear or branched alkyl groups having from 1 to 15 carbon            atoms;        -   alkyl groups comprising an alicyclic structure and having            from I to 15 carbon atoms;        -   aromatic groups having from 6 to 20 carbon atoms;        -   heteroaromatic groups having from 3 to 20 carbon atoms;    -   each Z is independently selected from the group consisting of:        -   —H        -   —C(O)OR^(CARB)        -   —C(═O)R^(ESTER)        -   —C(═O)NR^(A)R^(B)            wherein:    -   each R^(CARB) is organic groups comprising from 1 to about 20        carbon atoms;    -   each R^(ESTER) is organic groups comprising from 1 to about 20        carbon atoms;    -   each group —NR^(A)R^(B) is independently selected from the group        consisting of:        -   —NH₂        -   —NHR^(A)        -   —NR^(A)R^(B)        -   —NR^(AB)            wherein each monovalent R^(A) and R^(B) and each divalent            R^(AB) is independently an organic group comprising from 1            to 20 carbon atoms, and further comprising a reactive            conjugating functional group.

In one embodiment, said compound has the structure of Formula I. In oneembodiment, said compound has the structure of Formula II. In oneembodiment, said compound has the structure of Formula III. In oneembodiment, said compound has the structure of Formula IV. In oneembodiment, n is a positive integer from 2 to 4. In one embodiment, y¹,y², and y³ are each 2. In one embodiment, J is an oxygen atom. In oneembodiment, J is a covalent bond. In one embodiment, R^(C) is selectedfrom the group consisting of hydrocarbyl groups having from 1 to 20carbon atoms. In one embodiment, R^(C) is selected from the groupconsisting of:

In one embodiment, R^(C) is selected from the group consisting oforganic groups consisting only of carbon, oxygen, and hydrogen atoms,and having from 1 to 20 carbon atoms. In one embodiment, R^(C) is:

wherein p is a positive integer from 2 to 20. In one embodiment, eachG¹, G², and G³ is independently selected from the group consisting ofhydrocarbyl groups having from 1 to 20 carbon atoms. In one embodiment,each G¹, G², and G³ is —(CH₂)_(q)— wherein q is a positive integer from1 to 20. In one embodiment, each G¹, G², and G³ is independentlyselected from the group consisting of organic groups consisting only ofcarbon, oxygen, and hydrogen atoms, and having from 1 to 20 carbonatoms. In one embodiment, each G¹, G², and G³ is:

wherein p is a positive integer from 2 to 20. In one embodiment, R^(N)is independently selected from the group consisting of —H, —CH₃, and—CH₂CH₃. In one embodiment, each group —NR^(A)R^(B) is independentlyselected from the group consisting of:

Another aspect of the present invention pertains to a valency platformcompound having the structure of one of the following formulae:

wherein:

-   -   n is a positive integer from 1 to 10;    -   y¹, y², and y³ are independently a positive integer from 1 to        10;    -   J independently denotes either an oxygen atom or a covalent        bond;    -   R^(C) is selected from the group consisting of:        -   hydrocarbyl groups having from 1 to 20 carbon atoms;        -   organic groups consisting only of carbon, oxygen, and            hydrogen atoms, and having from 1 to 20 carbon atoms;        -   organic groups consisting only of carbon, oxygen, nitrogen,            and hydrogen atoms, and having from 1 to 20 carbon atoms;        -   organic groups consisting only of carbon, oxygen, sulfur,            and hydrogen atoms, and having from 1 to 20 carbon atoms;    -   each G¹, G², and G³ is independently selected from the group        consisting of:        -   hydrocarbyl groups having from 1 to 20 carbon atoms;        -   organic groups consisting only of carbon, oxygen, and            hydrogen atoms, and having from 1 to 20 carbon atoms;        -   organic groups consisting only of carbon, oxygen, nitrogen,            and hydrogen atoms, and having from 1 to 20 carbon atoms;    -   each R^(N) is independently selected from the group consisting        of:        -   hydrogen;        -   linear or branched alkyl groups having from 1 to 15 carbon            atoms;        -   alkyl groups comprising an alicyclic structure and having            from 1 to 15 carbon atoms;        -   aromatic groups having from 6 to 20 carbon atoms;        -   heteroaromatic groups having from 3 to 20 carbon atoms;    -   each Z is independently selected from the group consisting of:        -   —H        -   —C(═O)OR^(CARB)        -   —C(═O)R^(ESTER)        -   —C(═O)NR^(A)R^(B)            wherein:    -   each R^(CARB) is organic groups comprising from 1 to about 20        carbon atoms;    -   each R^(ESTER) is organic groups comprising from 1 to about 20        carbon atoms;    -   each group —NR^(A)R^(B) is independently selected from the group        consisting of:        -   —NH₂        -   —NHR^(A)        -   —NR^(A)R^(B)    -   wherein each monovalent R^(A) and R^(B) and each divalent R^(AB)        is independently an organic group comprising from 1 to 20 carbon        atoms, and further comprising a reactive conjugating functional        group.

In one embodiment, said compound has the structure of Formula V. In oneembodiment, said compound has the structure of Formula VI. In oneembodiment, said compound has the structure of Formula VII. In oneembodiment, n is a positive integer from 2 to 4. In one embodiment, y¹,y², and y³ are each 2. In one embodiment, J is an oxygen atom. In oneembodiment, J is a covalent bond. In one embodiment, R^(C) is selectedfrom the group consisting of hydrocarbyl groups having from 1 to 20carbon atoms. In one embodiment, R^(C) is selected from the groupconsisting of:

In one embodiment, R^(C) is selected from the group consisting oforganic groups consisting only of carbon, oxygen, and hydrogen atoms,and having from 1 to 20 carbon atoms. In one embodiment, R^(C) is:

wherein p is a positive integer from 2 to 20. In one embodiment, eachG¹, G², and G³ is independently selected from the group consisting ofhydrocarbyl groups having from 1 to 20 carbon atoms. In one embodiment,each G¹, G², and G³ is selected from the group consisting of:

In one embodiment, each G¹, G², and G³ is independently selected fromthe group consisting of organic groups consisting only of carbon,oxygen, and hydrogen atoms, and having from 1 to 20 carbon atoms. In oneembodiment, each R^(N) is independently selected from the groupconsisting of —H, —CH₃, and —CH₂CH₃. In one embodiment, each group—NR^(A)R^(B) is independently selected from the group consisting of:

Another aspect of the present invention pertains to methods of preparinga valency platform compound, as described herein.

Another aspect of the present invention pertains to a conjugatecomprising a valency platform compound, as described herein, covalentlylinked to one or more biologically active molecules. In one embodiment,said biologically active molecules are selected from the groupconsisting of: oligonucleotides, peptides, polypeptides, proteins,antibodies, saccharides, polysaccharides, epitopes, mimotopes, anddrugs.

Another aspect of the present invention pertains to methods of preparingconjugates, as described herein.

As will be appreciated by one of skill in the art, features of oneaspect or embodiment of the invention are also applicable to otheraspects or embodiments of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows certain valency platforms of the present invention,specifically, those having the structure of Formulae I, II, and III.

FIG. 2 shows certain valency platforms of the present invention,specifically, some of those having the structure of Formula I.

FIG. 3 shows certain valency platforms of the present invention,specifically, some of those having the structure of Formula I.

FIG. 4 shows certain valency platforms of the present invention,specifically, some of those having the structure of Formula II.

FIG. 5 shows certain valency platforms of the present invention,specifically, some of those having the structure of Formula II.

FIG. 6 shows certain valency platforms of the present invention,specifically, some of those having the structure of Formula III.

FIG. 7 shows certain valency platforms of the present invention,specifically, some of those having the structure of Formula I.

FIG. 8 shows certain valency platforms of the present invention,specifically, those having the structure of Formulae VI, V, and VI.

FIG. 9 shows a synthetic scheme for a simple example of “corepropagation” to obtain valency platforms of the present invention.

FIG. 10 shows synthetic schemes for the preparation of certainintermediates useful in the preparation of valency platforms of thepresent invention.

FIGS. 11A and 11B show synthetic schemes for the preparation of valencyplatform molecules of the present invention.

FIGS. 12A and 12B show synthetic schemes for the preparation of valencyplatform molecules of the present invention.

FIG. 13 shows a synthetic schemes for the preparation of valencyplatform molecules of the present invention.

FIGS. 14A and 14B show synthetic schemes for the preparation of valencyplatform molecules of the present invention.

FIG. 15 shows a synthetic schemes for the preparation of valencyplatform molecules of the present invention. FIGS. 16A and 16B showsynthetic schemes for the preparation of valency platform molecules ofthe present invention.

FIGS. 17A, 17B, 17C, and 17D show synthetic schemes for the preparationof valency platform molecules of the present invention.

FIGS. 18A and 18B show synthetic schemes for the preparation of valencyplatform molecules of the present invention.

FIG. 19 shows a synthetic schemes for the preparation of valencyplatform molecules of the present invention.

FIGS. 20A and 20B show synthetic schemes for the preparation of valencyplatform molecules of the present invention.

FIG. 21 shows the structure of examples of two carbamate compounds 39band 39c.

DETAILED DESCRIPTION OF THE INVENTION

Throughout this application, various publications, patents, andpublished patent applications are referred to by an identifyingcitation. The disclosures of the publications, patents, and publishedpatent specifications referenced in this application are herebyincorporated by reference into the present disclosure in their entirety.

In one embodiment, valency molecules are provided that comprisebranches, wherein at each branch, the molecule branches into two or morearms. The arms also may comprise branches. The valency molecule furthercomprises terminal groups on arms extending from the branches. Exemplaryterminal groups are reactive conjugating functional groups. This isillustrated in the Figures, for example, by compound 14 in FIG. 11B,which includes 6 branches and 8 terminal CBZ-protected amino groups.

Thus, in one embodiment, provided is a composition comprising valencymolecules, wherein each valency molecule comprises at least twobranches, at least four terminal groups, and at least 2 carbamatelinkages; and wherein said valency molecules have a polydispersity lessthan about 1.2, or for example, less than about 1.07. -The valencymolecules further can comprise, for example, at least 4 carbamatelinkages, at least 4 branches and at least 8 terminal groups. Thevalency molecules may be dendrimers.

The number of branches in the valency molecule may vary and may be, forexample, 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 16, 32, 64, 100 or morebranches. The number of branches can be, for example, 2-64, 2-32, 2-16,4-64, 4-32, 8-64, or 8-32. In a further embodiment, the number ofbranches may be, for example, at least 2, at least 4, at least 6, or atleast 8.

The number of carbamate linkages may vary. The valency molecule caninclude, for example, 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 15, 16, 18, 20,24, 29, 32, 64, 100 or more carbamate linkages. The number of carbamatelinkages can be, for example, 2-64, 2-32, 2-16, 4-64, 4-32, 8-64, or8-32. In a further embodiment, the number of carbamate linkages may be,for example, at least 2, at least 4, at least 6, or at least 8.

Each valency molecule can comprise for example, 1 to 100, e.g, 1-50terminal groups. For example, the valency molecule may comprise 4, 6, 8,9, 10, 12, 14, 15, 16, 18, 20, 21, 24, 29, or 32 or more terminalgroups. The valency molecule, for example, may comprise at least 4terminal groups, or at least 6 terminal groups, or at least 8 terminalgroups. The valency molecule in one embodiment has, for example, 4-16,4-32, 4-64, 8-32, 8-64, 12-32 or 12-64 terminal groups. The saidterminal groups are in one embodiment identical.

Examples of valency molecules include valency platform molecules.Valency molecules can be made as described herein for the synthesis ofvalency platform molecules.

A. VALENCY PLATFORMS

In one aspect, the present invention pertains to valency platformscomprising carbamate linkages and methods for the preparation of suchplatforms.

Particular advantages of the present invention include, but are notlimited to, (1) the ease of synthesis of valency platform molecules, (2)the metabolic stability of the carbamate linkages in the valencyplatform, (3) the ability to adjust the length and water solubility ofthe “arms” of the valency platform by using, for example, differentdialcoholamines, (4) the ability to further attenuate the properties ofthe valency platform by choice of the core group (e.g., attachment ofsolubilizing groups, chromophores, reporting groups, targeting groups,and the like).

In one embodiment, a composition is provided comprising valency platformmolecules, wherein each valency platform molecule comprises at least 2carbamate linkages and at least 4 reactive conjugating functionalgroups; and wherein said valency platform molecules have apolydispersity less than about 1.2, or optionally a polydispersity lessthan about 1.07. The valency platform molecules of the composition maycomprise, for example, at least 4 carbamate linkages and at least 8reactive functional groups. In one embodiment, the valency platformmolecules comprise at least 4 identical reactive conjugating functionalgroups. In another embodiment, the valency platform molecules comprise,for example, 2-32 carbamate linkages and 4-64 reactive functionalgroups. The valency platform molecules optionally may be linked to oneor more biologically active molecules, e.g., via the reactiveconjugating functional groups.

The valency molecules, such as valency platform molecules have theadvantage of having a substantially homogeneous (i.e., uniform)molecular weight (as opposed to polydisperse molecular weight), and arethus “chemically defined”. Accordingly, a population of these molecules(or conjugates thereof) are substantially monodisperse, i.e., have anarrow molecular weight distribution. A measure of the breadth ofdistribution of molecular weight of a sample of a platform molecule(such as a composition and/or population of platform molecules) is thepolydispersity of the sample. Polydispersity is used as a measure of themolecular weight homogeneity or nonhomogeneity of a polymer sample.Polydispersity is calculated by dividing the weight average molecularweight (Mw) by the number average molecular weight (Mn). The value ofMw/Mn is unity for a perfectly monodisperse polymer. Polydispersity(Mw/Mn) is measured by methods available in the art, such as gelpermeation chromatography. The polydispersity (Mw/Mn) of a sample ofvalency molecules is preferably less than 2, more preferably, less than1.5, or less than 1.2, less than 1.1, less than 1.07, less than 1.02,or, e.g., about 1.05 to 1.5 or about 1.05 to 1.2. Typical polymersgenerally have a polydispersity of 2-5, or in some cases, 20 or more.Advantages of the low polydispersity property of the valency platformmolecules include improved biocompatibility and bioavailability sincethe molecules are substantially homogeneous in size, and variations inbiological activity due to wide variations in molecular weight areminimized. The low polydispersity molecules thus are pharmaceuticallyoptimally formulated and easy to analyze.

Further there is controlled valency in a population of the valencymolecules. Thus, in a population of valency platform molecules, forexample, the number of attachment sites, e.g., reactive conjugatingfunctional groups, is controlled and defined. Each valency platformmolecule can comprise for example, 1 to 100, e.g, 1-50 attachment sites.For example, the valency platform molecule may comprise 4, 6, 8, 9, 10,12, 14, 15, 16, 18, 20, 21, 24, 29, or 32 or more attachment sites. Thevalency platform molecule, for example, may comprise at least 4attachment sites, or at least 6 attachment sites, or at least 8attachment sites. The valency platform molecule in one embodiment has,for example, 4-16, 4-32, 4-64, 8-32, 8-64, 12-32 or 12-64 attachmentsites. The said attachment sites are in one preferred embodimentidentical.

The number of carbamate linkages may vary. The valency platform moleculecan include, for example, 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 15, 16, 18,20, 24, 29, 32, 64, 100 or more carbamate linkages. The number ofcarbamate linkages can be, for example, 2-64, 2-32, 2-16, 4-64, 4-32,8-64, or 8-32. In a further embodiment, the number of carbamate linkagesmay be, for example, at least 2, at least 4, at least 6, or at least 8.

The valency platform molecule can comprise various combinations of thecarbamate linkages and attachment sites such as reactive functionalgroups depending on the method of preparation, for example, 2-32, e.g.,2-16 carbamate linkages; and 4-64, e.g., 4-32 reactive functionalgroups.

Formula I

In one embodiment, the present invention pertains to a valency platformhaving the structure of Formula I, as shown in FIG. 1.

In Formula I, n is a positive integer from 1 to 10, more preferably from1 to 5. In one embodiment, n is a positive integer from 2 to 10, morepreferably from 2 to 5. In one embodiment, n is 1. In one embodiment, nis 2. In one embodiment, n is 3. In one embodiment, n is 4.

In Formula I, y¹ is 1 or 2, and the subscript “2-y¹” is therefore 1 or0, respectively.

In Formula I, J independently denotes either an oxygen atom (i.e., —O—)or a covalent bond (i.e., no atom is present). When J is —O—, R^(C) isbound to the corresponding sidechain via a carbamate linkage (i.e.,—O—C(═O)—N<). When J is a covalent bond, R^(C) is bound to thecorresponding sidechain via an amide linkage (i.e., —C(═O)—N<).

In Formula I, R^(C) denotes a “core group,” that is, an organic groupwhich forms the core of the valency platform, and to which one or moresidechains is attached. The valency of the core group is determined byn. If n is 1, then R^(C) is monovalent; if n is 2, then R^(C) isdivalent; if n is 3, then R^(C) is trivalent; if n is 4, then R^(C) istetravalent, and so on.

In one embodiment, R^(C) is a hydrocarbyl group (i.e., consisting onlyof carbon and hydrogen) having from 1 to 20 carbon atoms, morepreferably from 1 to 10 carbon atoms, still more preferably from 1 to 6carbon atoms. In one embodiment, R^(C) is linear. In one embodiment,R^(C) is branched. In one embodiment, R^(C) comprises a cyclicstructure. In one embodiment, R^(C) is cyclic. In one embodiment, R^(C)is fully saturated. In one embodiment, R^(c) is partially unsaturated.In one embodiment, R^(C) comprises an aromatic structure. In oneembodiment, R^(C) is aromatic. In one embodiment, R^(C) is —CH₂—. In oneembodiment, R^(C) is —CH₂CH₂—. In one embodiment, R^(C) is —CH₂CH₂CH₂—.In one embodiment, R^(C) is:

In one embodiment, R^(C) is:

In one embodiment, R^(C) is an organic group consisting only of carbon,oxygen, and hydrogen atoms, and having from 1 to 20 carbon atoms, morepreferably from 1 to 10 carbon atoms, still more preferably from 1 to 6carbon atoms. In one embodiment, R^(C) is derived from a polyalkyleneoxide group. In one embodiment, R^(C) is derived from a polyethyleneoxide group. In one embodiment, R^(C) is a divalent polyalkylene oxidegroup. In one embodiment, R^(C) is a divalent polyethylene oxide group.In one embodiment, R^(C) is a divalent polypropylene oxide group. In oneembodiment, R^(C) is:

wherein p is a positive integer from 2 to about 200, more preferablyfrom 2 to about 50, more preferably from 2 to about 20, more preferablyfrom 2 to about 10, more preferably from 2 to about 6. In oneembodiment, p is 2. In one embodiment, p is 3. In one embodiment, p is4. In one embodiment, p is 5. In one embodiment, p is 6.

In one embodiment, R^(C) is an organic group consisting only of carbon,oxygen, nitrogen, and hydrogen atoms, and having from 1 to 20 carbonatoms, more preferably from 1 to 10 carbon atoms, still more preferablyfrom 1 to 6 carbon atoms. Examples of such core groups include, but arenot limited to, those derive from the “core compounds” described belowwhich consist only of carbon, oxygen, nitrogen, and hydrogen atoms.

In one embodiment, R^(C) is an organic group consisting only of carbon,oxygen, sulfur, and hydrogen atoms, and having from 1 to 20 carbonatoms, more preferably from 1 to 10 carbon atoms, still more preferablyfrom 1 to 6 carbon atoms. Examples of such core groups include, but arenot limited to, those derive from the “core compounds” described belowwhich consist only of carbon, oxygen, sulfur, and hydrogen atoms.

In Formula I, G¹ denotes an organic “linker group.” In one embodiment,G¹ is a hydrocarbyl group (i.e., consisting only of carbon and hydrogen)having from 1 to 20 carbon atoms, more preferably from 1 to 10 carbonatoms, still more preferably from 1 to 6 carbon atoms. In oneembodiment, G¹ is linear. In one embodiment, G¹ is branched. In oneembodiment, G¹ comprises a cyclic structure. In one embodiment, G¹ iscyclic. In one embodiment, G¹ is fully saturated. In one embodiment, G¹is partially unsaturated. In one embodiment, G¹ comprises an aromaticstructure. In one embodiment, G¹ is aromatic. In one embodiment, G¹ isdivalent. In one embodiment, R^(C) is —(CH₂)_(q)— wherein q is apositive integer from 1 to about 20, more preferably from 1 to about 10,more preferably from 1 to about 6, more preferably from 1 to about 4. Inone embodiment, G¹ is —CH₂—. In one embodiment, G¹ is —CH₂CH₂—. In oneembodiment, G¹ is —CH₂CH₂CH₂—.

In one embodiment, G¹ is an organic group consisting only of carbon,oxygen, and hydrogen atoms, and having from 1 to 20 carbon atoms, morepreferably from 1 to 10 carbon atoms, still more preferably from 1 to 6carbon atoms. In one embodiment, G¹ is derived from a polyalkylene oxidegroup. In one embodiment, G¹ is a divalent polyalkylene oxide group. Inone embodiment, G¹ is a divalent polyethylene oxide group. In oneembodiment, G¹ is a divalent polypropylene oxide group. In oneembodiment, G¹ is:

wherein p is a positive integer from 2 to about 200, more preferablyfrom 2 to about 50, more preferably from 2 to about 20, more preferablyfrom 2 to about 10, more preferably from 2 to about 6. In oneembodiment, p is 2. In one embodiment, p is 3. In one embodiment, p is4. In one embodiment, p is 5. In one embodiment, p is 6.

In one embodiment, G¹ is an organic group consisting only of carbon,oxygen, nitrogen, and hydrogen atoms, and having from 1 to 20 carbonatoms, more preferably from 1 to 10 carbon atoms, still more preferablyfrom 1 to 6 carbon atoms.

In Formula I, R^(N) denotes a nitrogen substituent, more specifically,an amino substituent. In one embodiment R^(N), if present, is hydrogen(i.e., —H). In one embodiment, R^(N), if present, is a linear orbranched alkyl group having from 1 to 15 carbon atoms, more preferablyfrom 1 to 10 carbon atoms, more preferably from 1 to 6 carbon atoms. Inone embodiment, R^(N), if present, is an alkyl group comprising analicyclic structure and having from 1 to 15 carbon atoms, morepreferably from 1 to 10 carbon atoms, more preferably from 1 to 6 carbonatoms. In one embodiment, R^(N), if present, is or comprises an aromaticgroup. In one embodiment, R^(N), if present, is or comprises aheteroaromatic group. In one embodiment, R^(N), if present, is orcomprises an aromatic group having from 6 to 20 carbon atoms, morepreferably from 6 to 15 carbon atoms, more preferably from 6 to 10carbon atoms. In one embodiment, R^(N), if present, is or comprises aheteroaromatic group having from 3 to 20 carbon atoms, more preferablyfrom 3 to 15 carbon atoms, more preferably from 3 to 10 carbon atoms. Inone embodiment, R^(N) is selected from the group consisting of —H, —CH₃,and —CH₂CH₃.

In Formula I, Z denotes a terminal group, which is independentlyselected from the group consisting of: —H (which yields a terminalalcohol group), —C(═O)OR^(CARB) (which yields a terminal carbonategroup), —C(═O)R^(ESTER) (which yield a terminal ester group), and—C(═O)NR^(A)R^(B) (which yields a terminal carbamate group).

In the above formulae, each R^(CARB) is a carbonate substituent or anactivated carbonate substituent. Many carbonate substituents are wellknown in the art, including, for example, organic groups comprising fromI to about 20 carbon atoms, including, for example, primary, secondary,and tertiary, substituted and unsubstituted, alkyl and aryl groupshaving from 1 to about 20 carbon atoms. Other examples of carbonategroups include those described herein for R^(N). Still other examples ofcarbonate groups include those described below for activated carbonates.

In the above formulae, each R^(ESTER) is an ester substituent or anactivated ester substituent. Many ester and activated ester substituentsare well known in the art, including, for example, organic groupscomprising from 1 to about 20 carbon atoms, including, for example,primary, secondary, and tertiary alkyl and aryl groups having from 1 toabout 20 carbon atoms. Other examples of carbonate groups include thosedescribed herein for R^(N). Examples of R^(ESTER) include, but are notlimited to, —CH₃ (to give an acetate group), —CH₂SH (to give amercaptoacetate group), and —CH₂C₆H₅, to give a benzoate group).

In one embodiment, Z is —NR^(A)R^(B) and denotes an amino group. Theamino group may be unsubstituted, in which case, R^(A) and R^(B) areboth hydrogen (i.e., —NR^(A)R^(B) is —NH₂). The amino group may bemonosubstituted, in which case R^(B) is hydrogen (i.e., —NR^(A)R^(B) is—NHR^(A)). The amino group may be disubstituted. In this case, R^(A) andR^(B) may be separate moieties, as in —NR^(A)R^(B), or R^(A) and R^(B)may be covalently linked together and form a divalent substituent,denoted R^(AB) (i.e., —NR^(A)R^(B) is —NR^(AB)). Thus, in oneembodiment, each group —NR^(A)R^(B) is independently selected from thegroup consisting of: —NH₂, NHR^(A), —NHR^(A)R^(B), and —NR^(AB), whereineach monovalent R^(A) and R^(B) and each divalent R^(AB) isindependently an organic group comprising from 1 to 20 carbon atoms, andfurther comprising a reactive conjugating functional group. In oneembodiment, each group —NR^(A)R^(B) is independently selected from thegroup consisting of: —NHR^(A), —NHR^(A)R^(B), and —NR^(AB). When nothydrogen, R^(A), R^(B), and R^(AB), preferably comprise a reactiveconjugating functional group.

The term “reactive conjugating functional group” is used herein to referto reactive functional groups which facilitate conjugation, for example,with a biologically active molecule. Examples of such reactiveconjugating functional groups include, but are not limited to, thefollowing:

In the above reactive conjugating functional groups, each X isindependently F, Cl, Br, I, or other good leaving group; each R^(ALK) isindependently an alkyl group, such as a linear or branched alkyl orcycloalkyl group having from 1 to about 20 carbon atoms; each R^(SUB) isindependently H or an organic group, such as a linear or branched alkylgroup, or a cycloalkyl group having from 1 to about 20 carbon atoms, anaryl group having from 6 to about 20 carbon atoms, or an alkaryl grouphaving from 7 to about 30 carbon atoms; each R^(ESTER) is independentlyan organic group having from 1 to about 20 carbon atoms, including, forexample, primary, secondary, and tertiary alkyl and aryl groups havingfrom 1 to about 20 carbon atoms; and, each R^(B) is independently aorganic group, such as an organic group comprising 1 to 50 atomsselected from the group consisting of C, H, N, O, Si, P, and S.

In a preferred embodiment, the reactive conjugating functional group isan amino group or a protected amino group. In one embodiment, the group—NR^(A)R^(B) comprises an amino group, and has the structure:

In one embodiment, the group —NR^(A)R^(B) comprises a protected aminogroup, and has the structure:

which is often conveniently abbreviated using “CBZ” to denote“carbobenzyloxy”:

In one embodiment, the group —NR^(A)R^(B) comprises a hydrobromide saltof an amino group, and has the structure:

In one embodiment, the group —NR^(A)R^(B) comprises a haloacetyl group(where X denotes Cl, Br, or I), and has the structure:

In one embodiment, the group —NR^(A)R^(B) comprises an amino group, andhas the structure:

wherein n is a positive integer from 1 to about 20, preferably from 1 toabout 10, preferably from 1 to about 5. In one embodiment, the group—NR^(A)R^(B) comprises a protected amino group, and has the structure:

which is often conveniently abbreviated using “BOC” to denote“tert-butoxycarbonyl”:

In one embodiment, the group —NR^(A)R^(B) comprises an amino group, andhas the structure:

wherein n is a positive integer from 1 to about 20, preferably from 1 toabout 10, preferably from 1 to about 5.

Examples of valency platforms having the structure of Formula I areshown in FIGS. 2, 3, and 7. In FIG. 2, the top structure has n=1 andy¹=1 and the bottom structure has n=2 and y¹=1. In FIG. 3, the topstructure has n=1 and y¹=2 and the bottom structure has n=2 and y¹=2. InFIG. 7, the structure has n=4 and y¹=2. The number of terminal groups—NR^(A)R^(B) is given by “n*y¹.” When “n*y¹” is 4, the structure mayconveniently be referred to as a “tetrameric” structure. When “n*y¹” is8, the structure may conveniently be referred to as a “octameric”structure. When “n*y¹” is 16, the structure may conveniently be referredto as a “hexadecameric” structure.

Examples of compounds having the structure of Formula I where Z is —Hinclude, but are not limited to, compounds 21, 24, 27a, 29, 32, and 38,described in the Examples below.

Examples of compounds having the structure of Formula I where Z is—C(═O)OR^(CARB) include, but are not limited to, compounds 22, 25, 27,30, 33, and 39 described in the Examples below.

Examples of compounds having the structure of Formula I where Z is—NR^(A)R^(B) include, but are not limited to, compounds 23, 23a, 26,26a, 31, 31 a, 34, 34a, 40, 41, 42, and 51, described in the Examplesbelow.

Formula II

In one embodiment, the present invention pertains to a valency platformhaving the structure of Formula II, as shown in FIG. 1.

In Formula II, n, R^(C), J, R^(A), R^(B), y¹, R^(N), G¹ , and Z are asdefined above for Formulae I. In Formula II, y² and G² are as definedabove for y¹ and G¹, respectively.

Examples of valency platforms having the structure of Formula II areshown in FIGS. 4 and 5. In FIG. 4, the top structure has n=1, y¹=2, andy²=1 and the bottom structure has n=1, y¹=2, and y²=2. In FIG. 5, thestructure has n=2, y¹=2, and y²=2. The number of terminal groups—NR^(A)R^(B) is given by “n*y¹*y².” When “n*y¹*y²” is 4, the structuremay conveniently be referred to as a “tetrameric” structure. When“n*y¹*y²” is 8, the structure may conveniently be referred to as a“octameric” structure. When “n*y¹*y²” is 16, the structure mayconveniently be referred to as a “hexadecameric” structure.

Examples of compounds having the structure of Formula II where Z is —Hinclude, but are not limited to, compounds 35, 43a, and 49a, describedin the Examples below.

Examples of compounds having the structure of Formula II where Z is.—C(═O)OR^(CARB) include, but are not limited to, compounds 35a, 43, and50, described in the Examples below.

Examples of compounds having the structure of Formula II where Z is—NR^(A)R^(B) include, but are not limited to, compounds 14, 15, 20, 20a,28, 28a, 36, 36a, 44, 44a, and 45, described in the Examples below.

Formula III

In one embodiment, the present invention pertains to a valency platformhaving the structure of Formula III, as shown in FIG. 1.

In Formula III, n, R^(C), J, R^(A), R^(B), y¹, y², R^(N), G¹, G^(2,) andZ are as defined above for Formulae I and II. In Formula III, y³ and G³are as defined above for y¹ and G¹, respectively.

Examples of a valency platform having the structure of Formula III isshown in FIG. 6. This structure has n=2, y¹=2, y²=2, and y³=2. Thenumber of terminal groups —NR^(A)R^(B) is given by “n*y¹*y^(2*y) ³.”When “n*y¹*y²*y³” is 4, the structure may conveniently be referred to asa “tetrameric” structure. When “n*y¹*y²*y³” is 8,the structure mayconveniently be referred to as a “octameric” structure. When“n*y¹*y²*y³” is 16, the structure may conveniently be referred to as a“hexadecameric” structure.

Formulae IV, V, and VI

In one embodiment, the present invention pertains to a valency platformhaving the structure of Formula IV, V, or VI, as shown in FIG. 8.

In these formulae, n, R^(C), J, R^(A), R^(B), R^(N), and Z are asdefined above for Formulae I through III. Unlike the compounds ofFormulae I through III, which may have branch points at nitrogen atoms,compounds of Formulae IV through VI may have branch points at a G group,for example, at G¹, G², or G³, and there may be one, two, three, or morebranches, for example, y¹, y², or y³ branches.

For Formulae IV through VII, G¹, G², and G³ are similar to G¹, G², andG³ for Formulae I through III. In a preferred embodiments, these groupsare trivalent, tetravalent or higher. In one embodiment, G¹, G², and G³are selected from the group consisting of:

Also, for Formulae IV through VI, y¹, y², and y³ are positive integersfrom 1 to about 10, more preferably from 1 to 5, more preferably from 1to 4, more preferably from 1 to 3, more preferably from 1 to 2.

Examples of compounds having the structure of Formula IV where Z is —Hinclude, but are not limited to, compound 46, described in the Examplesbelow.

Examples of compounds having the structure of Formula IV where Z is—C(═O)OR^(CARB) include, but are not limited to, compound 47, describedin the Examples below.

Examples of compounds having the structure of Formula V where Z is —Hinclude, but are not limited to, compound 47a, described in the Examplesbelow.

Examples of compounds having the structure of Formula V where Z is—C(═O)OR^(CARB) include, but are not limited to, compound 48, describedin the Examples below.

Examples of compounds having the structure of Formula V where Z is—NR^(A)R^(B) include, but are not limited to, compounds 48a, 48b, and48c, described in the Examples below.

In general, the number of termini may be calculated as the product of n,y¹, y², y³, etc., as discussed above. In one embodiment, this product is2 or more. In one embodiment, this product is more than 2. In oneembodiment, this product is more than 3. In one embodiment, this productis 4. In one embodiment, this product is 6. In one embodiment, thisproduct is 8. In one embodiment, this product is 16. In one embodiment,this product is 32.

In some embodiments, the valency platform molecule may be described as“dendritic,” owing to the presence of successive branch points.Dendritic valency platform molecules possess multiple termini, typically4 or more termini. In one embodiment, the valency platform molecule isdendritic and has 4 termini, such as, for example, compounds 23a, 26a,31 a, 34a, 42a, described in the examples below. In one embodiment, thevalency platform molecule is dendritic and has 8 termini, such as, forexample, compounds 15, 20a, 28a, 36a, 45, 48c and 51, described in theexamples below. In one embodiment, the valency platform molecule isdendritic and has 16 termini.

Note that Formulae I through VI are intended to encompass both“symmetric” and “non-symmetric” valency platforms. In one embodiment,the valency platform is symmetric. In one embodiment, the valencyplatform is non-symmetric. For example, each of the “n” groups which arependant from the core group, R^(C), may be the same or may beindependently different.

“Higher generation” valency platforms (e.g., 4th generation, 5thgeneration) are also contemplated, which have corresponding formulae.For example, 4th generation valency platforms would have G⁴ and y⁴, 5thgeneration valency platforms would further have G⁵ and y⁵, and so on forsuccessive generations. Also, “hybrid” valency platforms arecontemplated, which would include linkages of the sort found in FormulaeI through III as well as linkages of the sort found in Formulae IVthrough VI.

B. PREPARATION OF VALENCY PLATFORMS

In one embodiment, the valency platforms of the present invention may beprepared from “core” compounds which comprise one or more (say, j⁰)hydroxy groups (i.e., —OH). For example, the hydroxyl groups on the coreare converted to active carbonate derivatives, such as activatedcarbonate esters (for example, a para-nitrophenylcarbonate ester) andsubsequently reacted with a polyhydroxyamine compounds having j¹ hydroxygroups to provide a “first generation” carbamate with j¹ hydroxyl groupsfor each original hydroxyl group, for a total of j⁰*j¹ hydroxyl groups.The resulting hydroxy groups may then also be converted to activatedcarbonate derivatives, such as activated carbonate esters andsubsequently reacted with a polyhydroxyamine compound having j² hydroxygroups to provide a “second generation” carbamate with j² hydroxylgroups for each j¹ hydroxyl group, for a total of j⁰*j¹*j² hydroxylgroups. In this way, a dendritic structure may be constructed. Theprocess can be terminated at any “generation” by treating the terminalactivated carbonate derivatives, such as activated carbonate esters,with an appropriately functionalized compound (for example, anmono-protected diamine) to provide whatever functionality is desired atthe termini.

In one embodiment, the valency platforms of the present invention may beprepared from a “segmental approach” in which “segments” areindependently synthesized and subsequently attached to a “core” group.

In another embodiment, an alternative, more efficient “core propagation”process has been developed in which a core group is modified in aniterative process to generate a dendritic structure. The corepropagation approach involves fewer steps and is preferred over thesegmental approach.

In another embodiment, the valency platforms of the present inventionmay be prepared using solid phase synthesis from a hydroxyl containingresin. Such an embodiment is illustrated in FIG. 20. A hydroxyl groupattached to a solid phase by a cleavable linker provides a way ofbuilding a dendrimeric scaffold using solid phase synthesis. The abilityto prepare scaffolds on the solid phase can be particularly useful forthe rapid synthesis of dendrimeric platforms with minimal purification.Also, solid phase dendrimeric platforms can be used to generatecombinatorial libraries of multivalent compounds.

In one preferred “core propagation” approach, the synthesis typicallybegins with an alcohol containing “core compound.” In principle, anyhydroxyl-containing compound can be used. Examples of alcohol containing“core compounds” having one hydroxyl group (i.e., —OH) include, but arenot limited to:

Other examples of alcohol containing “core compounds” having onehydroxyl group (i.e., —OH) include, but are not limited to,mono-hydroxylamines, such as those described below, for which the aminogroup may be in a protected form, for example, using a BOC or CBZprotecting group.

Examples of alcohol containing “core compounds” having two hydroxylgroups (i.e., —OH) include, but are not limited to:

Other examples of alcohol containing “core compounds” having twohydroxyl groups (i.e., —OH) include, but are not limited to, primary orsecondary amines having two hydroxyl groups, such as those describedbelow. Again, the amino group may be in a protected form, for example,using a BOC or CBZ protecting group.

Examples of alcohol containing “core compounds” having three hydroxylgroups (i.e., —OH) include, but are not limited to:

Other examples of alcohol containing “core compounds” having three ormore hydroxyl groups (i.e., —OH) include, but are not limited to,primary or secondary amines having three hydroxyl groups, such as thosedescribed below. Again, the amino group may be in a protected form, forexample, using a BOC or CBZ protecting group.

Examples of alcohol containing “core compounds” having four hydroxylgroups (i.e., —OH) include, but are not limited to:

Further examples of alcohol containing “core compounds” include, but arenot limited to, those which comprise a sulfhydryl group (i.e., —SH),which may be protected, for example, with a trityl protecting group(i.e., as —S-Tr, that is, —S—C(C₆H₅)₃) or as a disulfide (i.e., as—S—SR). Examples of core groups which have a protected sulfhydryl groupinclude, but are not limited to, the following:

where n is from 1 to about 200, preferably from 1 to about 20.

In addition, hydroxyl groups on solid phase synthesis resins can be usedas core groups to provide dendrimeric carbamate residues on solid phasewhich can be used to boost the valence of the resin or cleaved off theresin. For example, a Wang resin of the following form may be used:

In one embodiment, a hydroxy containing core group may be prepared froma corresponding carboxylic acid compound or halocarbonyl compound:

Core: compounds which possess-amino or sulfhydryl groups, which may beprotected or unprotected, may be used to covalently attach the resultingvalency platform molecule to other molecules of interest, via the coregroup rather than via the termini using conjugation methods such asthose described herein.

In one step, the hydroxyl groups of the alcohol containing core groupare converted to active carbonate derivatives. The active carbonatederivative in one embodiment has the formula:

where X is a leaving group such as Cl, imidazole or thiolate.

The hydroxyl groups of the alcohol can be converted to active activecarbonates by reaction of the hydroxyl groups of the alcohol containingcore group with a phosgene equivalent. Phosgene equivalents withappropriate reactivity can be selected. The phosgene equivalent has, forexample, the structure X₁(CO)X₂ where X₁ and X₂ are both leaving groups.X₁ and X₂ each independently can be chosen from typical leaving groupsin acylation chemistry such as alkoxide, thiolate, halide, andimidazole. In one preferred embodiment, the phosgene equivalent is4-nitrophenylchloroformate. In another embodiment, the phosgeneequivalent is carbonyldiimidazole. Other exemplary phosgeneiequivalentsinclude phosgene, N,N′-succinimidylcarbonate, succinimidyl2,2,2-trichloroethylcarbonate, bis-4-nitrophenylcarbonate, triphosgene,2,2,2-trichloroethylchloroformate, 4-nitrophenylchloroformate,phenylchloroformate, N-hydroxysuccinimidylchloroformate,trichloromethylchloroformate, ethylchlorothiolformate,di-(1-benzotriazolyl)carbonate, and 4-nitrophenylsuccinimidylcarbonate.

Thus, in one embodiment, to form an active carbonate derivative in thesynthesis of the valency platform molecule, an alcohol is reacted withthe phosgene equivalent to form the activated carbonate by displacingX₁. X₁ is chloride in one preferred embodiment. The active carbonatederivative is used to acylate an aminoalcohol on the nitrogen, formingthe carbamate bond, then the phosgene again is added to convert thehydroxyl group to another active carbonate derivative. An example of anactive carbonate derivative is compound 39c shown in FIG. 21.

In one embodiment, the active carbonate derivative is a carbonate ester.The terms “carbonate” and “carbonate ester” are used herein in theconventional sense and relate to species which comprise the followingstructure:

wherein R¹ denotes a carbonate group, such as an organic group havingfrom 1 to 20 carbon atoms. The terms “activated carbonate” and“activated carbonate ester” are used herein to refer to carbonates forwhich R¹ is an activating group, and for which the moiety —O—R¹ forms agood leaving group. A particularly preferred class of activatedcarbonate esters include, but are not limited to para-nitrophenylcarbonate ester compounds of the formula:

Such “PNP” activated carbonate esters may readily be formed from thecorresponding alcohol, R—OH by reaction with PNP chloroformate in thepresence of pyridine (C₅H₅N) in methylene chloride (CH₂Cl₂).

Examples of other activated carbonate groups include, but are notlimited to, the following:

In another step, the activated carbonate ester is converted to thecorresponding carbamate. The above PNP activated carbonate esters arereadily converted to the corresponding carbamates by reaction with anamine. The dendritic structure may be extended by employing a primary orsecondary amine having j¹ hydroxy groups. In this way, each originalhydroxy group, which led to an activated carbonate ester group, thenleads to j¹ hydroxy groups.

For example, the PNP activated carbonate ester may be reacted with aprimary or secondary dihydroxyamine. Examples of primary and secondaryamines having two hydroxyl groups include, but are not limited to:

In one step, the activated carbonate ester is converted to thecorresponding carbamate using a monohydroxyamine to maintain valencyfrom one generation to the next yet impart unique properties such as armlength, steric bulk, solubility, or other physical properties. Examplesof such monohydroxyamines include, but are not limited to:

In one step, the activated carbonate ester is converted to thecorresponding carbamate using a primary or secondary amine which acts asa “terminating” amine. In one embodiment, a mono-protected diamine isemployed. In a preferred embodiment, the terminating amine is a mono-CBZprotected piperazine, since this compound provides a convenientsecondary amine handle for adding functionality by acylation with otherreactive groups such as haloacetyl, maleimidoyl, etc. depending on whatis desired at the N-terminus. For example, reaction withmono-CBZ-protected piperazine in the presence of triethylamine((CH₃CH₂)₃N) in methylene chloride (CH₂Cl₂) yields the CBZ-protectedpiperazine carbamate, which can then be converted to a haloacetyl group:

Ethylenediamine and other diamines can function similarly.. Examples ofpreferred terminating amines include, but are not limited to those shownbelow, as well as mono-protected (e.g., mono-CBZ-protected) formsthereof:

A particularly preferred terminating amines is mono-CBZ-protectedpiperazine:

In principle any primary or secondary amine containing compound whichcontains a reactive conjugating group (such as those described above) ora biologically active molecule can be used to terminate the dendrimerand provide the terminal functionality that is desired. For example,amino alcohols would provide terminal hydroxyl groups, amino aldehydeswould provide terminal aldehyde groups, amino acids would provideterminal carboxylic acids, and aminothiols would provide terminalthiols. Methods for the introduction of other reactive conjugatingfunctional groups, such as those described above, as terminating groupsare well known to those of skill in the art.

C. VALENCY PLATFORM CONJUGATES, METHODS OF PREPARATION, AND USES THEREOF

In one embodiment, valency platform molecules are provided which act asscaffolds to which one or more molecules may be covalently tethered toform a conjugate. Thus, in another aspect, the present inventionpertains to valency platform conjugates.

In one embodiment, the valency platform; is covalently linked to one ormore biologically active molecules, to form a conjugate. The term“biologically active molecule” is used herein to refer to moleculeswhich have biological activity, preferably in vivo. In one embodiment,the biologically active molecule is one which interacts specificallywith receptor proteins.

In one embodiment, the valency platform is covalently linked to one ormore oligonucleotides, to form a conjugate. In one embodiment, thevalency platform is covalently linked to one or more peptides, to form aconjugate. In one embodiment, the valency platform is covalently linkedto one or more polypeptides, to form a conjugate. In one embodiment, thevalency platform is covalently linked to one or more proteins, to form aconjugate. In one embodiment, the valency platform is covalently linkedto one or more antibodies, to form a conjugate. In one embodiment, thevalency platform is covalently linked to one or more saccharides, toform a conjugate. In one embodiment, the valency platform is covalentlylinked to one or more polysaccharides, to form a conjugate. In oneembodiment, the valency platform is covalently linked to one or moreepitopes, to form a conjugate. In one embodiment, the valency platformis covalently linked to one or more mimotopes, to form a conjugate. Inone embodiment, the valency platform is covalently linked to one or moredrugs, to form a conjugate.

In one embodiment, the biological molecule is first modified to possessa functionalized linker arm, to facilitate conjugation. An example ofsuch a functionalized linker arm is a polyethylene glycol disulfide,such as, for example:

One advantage of the valency platforms of the present invention is theability to introduce enhanced affinity of the tethered biologicallyactive molecules for their binding partners. Another advantage of thevalency platforms of the present invention is the ability to facilitatecrosslinking of multiple ligands,,as is useful in B cell tolerance.Another advantage of the valency platforms of the invention is theability to include functionality on the “core” that can be independentlymodified to enable the preparation of conjugates which can be tailoredfor specific purposes.

Conjugates of the valency platform molecule and one or more biologicallyactive molecules may be prepared using known chemical synthetic methods.As discussed above, the termini of the valency platform molecule (i.e.,the R^(A), R^(B), and/or R^(AB) of the group —NRAR^(B), as discussedabove) preferably comprise a reactive conjugating functional group, andthis reactive functional group may be used to couple the valencyplatform to the desired biologically active molecule.

In one embodiment, the reactive haloacetyl group may be used to couplethe valency platform to a biologically active molecule which possessesone or more reactive conjugating functional groups which are reactivetowards the haloacetyl group, and which react to yield a covalentlinkage.

For example, if the biologically active molecule is a protein which hasone or more free amino groups (i.e., —NH₂), the two groups may be usedto form the conjugate:

In another example, if the biologically active molecule is a proteinwhich has one or more free thiol groups (i.e., —SH) or sulfide groups(ie., —SR), the two groups may be used to form the conjugate:

In another embodiment, a terminal maleimidoyl group may be used tocouple the valency platform to a biologically active molecule whichpossesses one or more reactive conjugating functional groups which arereactive towards the maleimidoyl group, and which react to yield acovalent linkage.

For example, if the biologically active molecule is a protein which hasone or more free thiol groups (i.e., —SH) or sulfide groups (i.e..,—SR), the two groups may be used to form the conjugate:

D. EXAMPLES

Several embodiments of the present invention are illustrated in theExamples below, which are offered by way of illustration and not by wayof limitation.

Example 1

Examples of Synthesis of Amine Diols

A chemical scheme for the preparation of HEGA(bis-hexaethyleneglycolamine) is shown in FIG. 10. One hydroxy terminusof hexaethylene glycol is first converted to a tosyl group (compound ),which is then converted to a bromo group (compound 2). The resultingcompound is then reacted with tosylamide to yield tosylatedbis-hexaethyleneglycolamine (compound 3). The tosyl group is thenremoved to yield the desired bis-hexaethyleneglycolamine (compound 4).

A chemical scheme for the preparation of DEGA(bis-diethyleneglycolamine) is also shown in FIG. 10. Chlorodiethyleneglycol (compound 5) is reacted with aminodiethylene glycol (compound 6)to yield the desired bis-diethyleneglycolamine (compound @

Compound 1 Hexaethyleneglycol mono-tosylate

25 g (88.5 mmol) of hexaethyleneglycol was stirred at. 0° C. in 200 mLof CH₂Cl₂, and 14.3 mL of pyridine (177 mmol; 2 eq.) was added to themixture followed by 17.4 g (88.5 mmol) of tosylchloride. The reactionmixture was stirred at room temperature for 24 hours and partitionedbetween 400 ml of 1N HCl and 200 ml of CH₂Cl₂. The organic layer wasdried over MgSO₄, filtered, and concentrated to provide 31 g of a lightyellow oil. Purification by silica gel chromatography (CH₂Cl₂/MeOH)provided 15.32 g (40%) of 1 as a light yellow oil: ¹H NMR (CDCl₃) δ 2.45(s, 3H), 3.55-3.75 (m, 22H), 4.15 (t, 2H), 7.35 (d, 2H), 7.80 (d, 2H);HRMS (FAB) calculated for C₁₉H₃₃O₉S (M+H): 437.1845. Found: 437.1834.

Compound 1 Hexaethyleneglycol mono-tosylate

50 g of HEG (177 mmol) was dissolved in 300 ml of CH₂Cl₂ and 7.2 ml (88mmol) of pyridine was added at room temperature. 17.4 g (88 mmol) oftosylchloride was added to the mixture in four batches, each 2 hoursapart. After the last addition, the mixture was stirred for 16 hours.The reaction mixture was concentrated, 150 mL of 0.1 M HCl was added,and the mixture was extracted twice with hexane to remove excesstosylchloride. The aqueous layer was washed with three portions of etherto remove di-tosylate. This was carefully monitored by TLC to avoid anyremoval of mono-tosylate. The aqueous layer was then extracted withportions of CH₂Cl₂. The combined organic layers were washed with 0.1 MHCl, dried over MgSO₄, filtered, and concentrated to give 23.6 g (31%)of compound 1.

Compound 2 Hexaethyleneglycol mono-bromide

Compound 1 (18 g, 41.3 mmol) was dissolved in 120 mL of acetone and 10.8g of LiBr (124 mmol) was added. The mixture was stirred at 60° C. for 2hours, the reaction mixture was allowed to cool to room temperature, and500 mL of H₂O was added. The mixture was extracted with 2×500 mL ofCH₂Cl₂. The combined organic layers were dried over MgSO₄, filtered, andconcentrated to give 13.7 g (96%) of compound 2 as a light yellow oil:¹H NMR (CDCl₃) δ 3.50 (t, 2H), 3.60-3.75 (m, 20H), 3.83 (t, 2H); HRMS(FAB) calculated for C₁₂H₂₆BrO₆ (M+H): 345.0913. Found: 345.0922.

Compound 3 N,N-bis-hexethyleneglycol-tosylamide

Compound 2 (3.5 g, 9.8 mmol) and 0.84 g (4.9 mmol) of tosylamide weredissolved in 35 mL of CH₃CN. Potassium carbonate (1.63 g (11.8 mmol),which had been dried in the 100° C. oven, was added, and the mixture wasrefluxed for 18 hours under N₂. The mixture was allowed to cool to roomtemperature, and 150 mL of H₂O was added. The mixture was extracted with3×150 mL of CH₂Cl₂. The combined organic layers were washed with H₂O,dried (MgSO₄), filtered, and concentrated to give 3.3 g of alight yellowoil. Purification by silica gel chromatography (CH₂Cl₂ /MeOH) provided2.7 g(79%) of compound 3 as a light yellow oil: ¹H NMR (CDCl₃) δ 2.45(s, 3H), 3.35 (t, 4H), 3.55-3.8 (m, 44H), 7.27 (d, 2H), 7.69, (d, 2H);HRMS (FAB) calculated for C₃₁H₅₇CsNO₁₄S (M+Cs): 832.2554. Found:832.2584.

Compound 4 N,N-bis-hexethyleneglycol-amine

Compound 3, (2.74 g) was dissolved in 4 mL of dry THF and transferred toa three-neck flask equipped with a Dewar-condenser. This was stirred at−78° C. as 100 mL of NH₃ was condensed into the mixture. Approximately1-2 g of Na was added to the mixture at −78° C. in small portions untilthe dark blue color persisted. The cooling bath was removed, and themixture was then stirred at reflux for 30 minutes. Cooling at −78° C.was continued, and the reaction was carefully quenched with glacialacetic acid until all the blue color disappeared. The NH₃ was allowed toevaporate, and the white solid was dried under vacuum to yield compound4. This material was used as is in subsequent steps assuming a 100%yield.

Compound 7 DEGA (bis-diethyleneglycolamine)

Compound 7 was prepared according to an existing literature procedure asshown below (Bondunov et al., J. Org. Chem. 1995, Vol. 60, pp.6097-6102). 33.8 g (321 mmol) of aminodiethyleneglycol (compound 6), 9.4g ( 88.3 mmol) of Na₂CO₃, 200 mL of toluene, and 10.0 g (80.3 mmol) ofchlorotriethyleneglycol (compound 5), were refluxed with a Dean-Starketrap to remove water for 48 hours. The mixture was allowed to cool thenfiltered and concentrated. The resulting 45.8 g of material was vacuumdistilled (bp 153-158° C., 0.1 Torr) to yield 12.0 g (78%) of compound7.

Example 2

Synthesis of Octamer of HEGA/TEG Using Segmental Approach

A chemical scheme for the preparation of an octamer of HEGA/TEG is shownin FIGS. 11A and 11B. Compound. The bis-hexaethyleneglycolamine(compound 4) was reacted with di-tert-butyldicarbonate to yield theN-BOC compound (compound 8), which was then reacted withpara-nitrophenylchloroformate to yield the para-nitrophenylcarbonatecompound (compound 9). The para-nitrophenylcarbonate (PNP) group wasthen converted to a carbamate group by reaction with mono-CBZ-protectedpiperazine, yielding compound 10. The BOC group was removed usingtrifluoroacetic acid to yield compound 11. Compounds 9 and 11 were thenreacted together to form a “one-sided” dendritic compound (compound 12).Again, the BOC group was removed using trifluoroacetic acid to yieldcompound 13. Compound 13 was then reacted with triethyleneglycol bischloroformate (from which the “core” is derived) to yield the“two-sided” dendritic compound (compound 14). The terminal CBZ-protectedamino groups were then converted to the hydrobromide salt of aminogroup, and further reacted with bromoacetic anhydride to yield reactivebromoacetyl groups at each of the termini in compound 15.

Compound 8 N-BOC-N,N-bis-hexaethyleneglycol-amine

Compound 4 (797 mg, 1.46 mmol) was dissolved in 14 mL of H₂O, and themixture was stirred at room temperature. To the mixture was added 465 mg(4.38 mmol) of Na₂CO₃. The pH was checked to make sure it was basic, and319 mg (1.46 mmol) of di-tert-butyldicarbonate ((BOC)₂O)was dissolved in7 mL of dioxane and the resulting solution was added to the reactionmixture. The mixture was stirred at room temperature. for 6 h andpartitioned between 100 ml of H₂O and 3×100 mL of CH₂Cl₂. The combinedorganic layers were dried (MgSO₄), filtered, and concentrated to give605 mg (64%) of compound 8 as a light yellow oil: ¹H NMR (CDCl₃) δ 1.45(s, 9H), 3.45 (m, 4H), 3.8-3.5 (m, 44H): MS (ESI) calculated forC₂₉H₅₉NaNO₁₄ (M+Na): 668. Found: 668.

Compound 9

Compound 8 (52 mg, 0.08 mmol) was dissolved in 3 mL of CH₂Cl₂ and 97 mg(0.483 mmol) of p-nitrophenylchloroformate was added to the mixture. Themixture was stirred at 0° C., 78 μL (0.966 mmol) of pyridine was added,and the mixture was then stirred at room temperature for 4 hours. Thereaction mixture was cooled to 0° C., acidified with 1 N HCl, andpartitioned between 10 mL of 1 N HCl and 3×10 mL of CH₂Cl₂. The combinedorganic layers were dried (MgSO₄), and concentrated to give 132 mg of anoil. Purification was accomplished by silica gel chromatography (98:2CH₂Cl₂/MeOH) to give 57 mg (74.0%) of compound 9 as an oil: ¹H NMR(CDCl₃) δ 1.49 (s, 9H), 3.43 (m, 4H), 3.52-3.77 (m, 36H), 3.83 (m, 4H),4.47 (m, 4H), 7.41 (d, 4H), 8.32 (d, 4H); MS (ESI) calculated forC₄₃H₆₅NaN₃O₂₂ (M+Na): 998. Found: 998.

Compound 10

Compound 9 (2.72 g, 2.79 mmol) was dissolved in 10 mL of CH₂Cl₂ and themixture was stirred at 0° C. To the mixture was added 1.16 ml (8.36mmol) of Et₃N followed by 1.842 g (8.36 mmol) of mono-CBZ-piperazinedissolved in 10 mL of CH₂Cl₂. The mixture was stirred at roomtemperature for 18 hours, cooled to 0° C., and acidified with 1 N HCl.The mixture was partitioned between 150 mL of 1 N HCl and 3×150 mL ofCH₂Cl₂. The combined organic layers were washed with saturated NaHCO₃solution, dried (MgSO₄) and concentrated to give 3.64 g of a yellow oil.Purification by silica gel chromatography (97/3 CH₂Cl₂/MeOH) gave 3.1 g(98%) of compound 10 as a light yellow oil: ¹H NMR (CDCl₃) δ 1.45 (s,9H), 3.40-3.55 (m, 12H), 3.55-3.68 (m, 36H), 3.71 (m, 4H), 4.37 (m, 4H),5.17 (s, 4H), 7.35 (brd s, 10H); MS (ESI) calculated for C₅₅H₈₇NaN₅O₂₀(M+Na): 1060. Found: 1060.

Compound 11

Compound 10 (3.54 g, 3.11 mmol) was dissolved in 15 mL of CH₂Cl₂ and 15mL of trifluoroacetic acid (TFA) was added to the mixture. The mixturewas stirred at room temperature for 4 hours and concentrated. Theresidue was re-dissolved in 10 m]L of CH₂Cl₂ and neutralized by shakingwith a saturated solution of NaHCO₃ at 0° C. The mixture was thenpartitioned between 100 mL of saturated NaHCO₃ solution and 4×100 mL ofCH₂Cl₂. The combined organic layers were dried (MgSO₄), filtered, andconcentrated to give 3.16 g of compound 11 (98%) as a yellow oil: ¹HNMR(CDCl₃) δ 2.88 (t, 4H), 3.50 (brd s, 16H), 3.56-3.69 (m, 36H), 3.71(m, 4H), 4.28 (m, 4H), 5.15 (s, 4H), 7.38 (brd s, 10H); MS (ESI)calculated for C₅₀H₈₀N₅O₁₈ (M+H): 1037. Found: 1038.

Compound 12

Compound 9 (647 mg, 0.663 mmol) and 2.065 g (1.989 mmol) of compound 11were dissolved in 3 mL of CH₂Cl₂, and 462 μL (3.315 mmol) of Et₃N and 40mg (0.331 mmol) of DMAP (4-dimethylaminopyridine) was added to themixture. The reaction mixture was stirred at room temperature overnightand cooled to 0° C. To the mixture was added 5 mL of H₂O, and themixture was acidified with 1 N HCl and partitioned between 50 mL of H₂Oand 3×50 mL of CH₂Cl₂. The combined organic layers were dried (MgSO₄)and concentrated to give 2.77 g (72.1%) of a yellow oil. Purification bysilica gel chromatography (CH₂Cl₂/MeOH) gave 1.307 g (72%) of compound12 as a yellow oil: ¹H NMR (CDCl₃) δ 1.46 (s, 9H), 3.49 (brd s, 32H),3.52-3.67 (m, 92H), 3.69 (m, 8H), 4.22 (m, 12H), 5.15 (s, 8H), 7.35 (brds, 20H).

Compound 13

Compound 12 (1.3 g, 0.47 mmol) of the starting material was dissolved in10 mL of CH₂Cl₂ and 10 mL of TFA (trifluoroacetic acid) was added. Themixture was stirred at room temperature for 4 hours, concentrated, andre-dissolved in 10 mL of CH₂Cl₂. The mixture was neutralized shakingwith a saturated solution of NaHCO₃ at 0° C. The mixture was partitionedbetween 25 mL of saturated NaHCO₃ solution and 4×25 mL of CH₂Cl₂. Thecombined organic layers were dried (MgSO₄) and concentrated to give 1.23g (98%) of compound 13 as a yellow oil: ¹H NMR (CDCl₃) δ 3.48 (brd s,32H), 3.53-3.67 (m, 92H), 3.71 (m, 8H), 4.22 (m, 12H), 5.15 (s, 8H),7.38 (brd s, 20H).

Compound 14

Compound 13 (600 mg, 0.224 mmol) was dissolved in 1.5 mL of CH₂Cl₂ andthe mixture was stirred at 0° C. To the mixture was added 65 μL (0.373mmol) of DIPEA followed by the slow addition of a solution of 20.5 mg(0.075 mmol) of triethyleneglycol bis-chloroformate dissolved in 0.5 mLof CH₂Cl₂. After 3 hours the reaction mixture was cooled to 0° C. andacidified with 1 N HCl. The mixture was partitioned between 25 ml of 1 NHCl and 2×25 ml of CH₂Cl₂. The combined organic layers were dried(MgSO₄) and concentrated to give 566 mg of a light yellow oil.Purification by silica gel chromatography (95/5 CH₂Cl₂/MeOH) gave 145 mgof compound 14 (35%) as a light yellow oil: ¹H NMR (CDCl₃) δ 3.51 (brds, 64H), 3.54-3.77 (m, 272H), 4.23 (m, 28H), 5.17 (s, 16H), 7.36 (brd s,40H); MS (ESI) calculated for C₂₆₀H₄₂₁N₂₂O₁₀₆ (M+H): 5549. Found: 5549.

Compound 15

Compound 14 (143 mg, 0.026 mmol) was treated with 3 mL of 30% HBr/AcOHfor 30 min. The resulting HBr salt was precipitated with ether. Thesolids were collected by centrifugation and washed three times withether. The resulting HBr salt was dried in the desiccator overnight anddissolved in 1.2 ml of H₂O. The mixture was stirred at 0° C., and 97 mg(1.16 mmol) of sodium bicarbonate was added. A solution of 107 mg (0.412mmol) of bromoacetic anhydride in 1.2 mL of dioxane was added, themixture was stirred at 0° C. for 15-20 min. To the mixture was added 10mL of H₂O, and the mixture was slowly acidified with 1 M H₂SO₄ to a pHof 4. The aqueous layer was extracted with 2×10 mL of EtOAc which wasdiscarded. The aqueous layer was then extracted with 6×10 mL of 8/2CH₂Cl₂/MeOH. The combined organic layers were dried (MgSO₄), filteredand concentrated to give 117 mg of an oil. Preparative HPLC (C18,gradient, 35-55% B, A=0.1% TFA/H₂O and B=0.1% TFA/CH₃CN) to give 27 mg(19%) of compound 15 as a colorless oil: ¹H NMR (CDCl₃) 3.46-3.75 (m,336H), 3.90 (m, 16H), 4.21-4.33 (m, 28H); MS (ESI) calculated forC₂₁₂H₃₈₁Br₈N₂₂O₉₈ (M+H): 5435. Found: 5448.

Example 3

Synthesis of Octamer of DEGA/TEG Using Segmental Approach

A chemical scheme for the preparation of an octamer of DEGA/TEG is shownin FIGS. 12A and 12B. The bis-diethyleneglycolamine (compound 7) wasreacted with di-tert-butyldicarbonate to yield the N-BOC compound(compound 16), which was then reacted with para-nitrophenylchloroformateto yield the para-nitrophenylcarbonate compound (compound 17). Thepara-nitrophenylcarbonate (PNP) group was then converted to a carbamategroup by reaction with mono-CBZ-protected piperazine, yielding compound18. The BOC group was removed using trifluoroacetic acid to yieldcompound 18a. Compounds 17 and 18a were then reacted together to form a“one-sided” dendritic compound (compound 19). Again, the BOC group wasremoved using trifluoroacetic acid to yield compound 19a. Compound 19awas then reacted with triethyleneglycol bis chloroformate (from whichthe “core” is derived) to yield the “two-sided” dendritic compound(compound 20). The terminal CBZ-protected amino groups were thenconverted to the hydrobromide salt of amino group, and further reactedwith bromoacetic anhydride to yield reactive bromoacetyl groups at eachof the termini in compound 20a.

Compound 16 N-BOC-N,N-bis-diethyleneglycol-amine

To a solution of 600 mg ( 3.10 mmol) of bis-diethyleneglycolamine(compound 7) in 9.9 mL of 10% aqueous Na₂CO₃ was added slowly a solutionof 678 mg (3.10 mmol) of di-tert-butyldicarbonate in 5 mL of dioxane.The mixture was stirred at room temperature for 5 hours, and 25 mL ofwater was added. The mixture was shaken with Et₂O, the Et₂O layer wasdiscarded, and the mixture was extracted with three 25 mL portions ofCH₂Cl₂. The CH₂Cl₂ extracts were combined, dried (MgSO₄), filtered andconcentrated to give 717 mg (79%) of compound 16 as a viscous oil: ¹HNMR (CDCl₃) δ 1.48 (s, 9H), 3.45 (brd s, 4H), 3.60 (t, 4H), 3.65 (brd s,4H), 3.69 (brd s, 4H).

Compound 17

To a solution of 200 mg (0.68 mmol) of compound 16 and 822 mg g (4.08mmol) of 4-nitrophenylchloroformate in 30 mL of CH₂Cl₂ was added 0.66 mL(8.16 mmol) of pyridine at 0° C. The mixture was stirred at roomtemperature for 1.5 hours then cooled back to 0° C., and the mixture wasacidified with 1 N HCl and partitioned between 50 mL of 1 N HCl andthree 50 mL portions of CH₂Cl₂. The combined CH₂Cl₂ extracts were dried(MgSO₄), filtered and concentrated to give 1.21 g of yellow oil. Themixture was partially purified by silica gel chromatography(CH₂Cl₂/MeOH) to give 581 mg of partially purified compound 17 whichstill contained 4-nitrophenol: MS (ESI) calculated for C₂₇H₃₃NaN₃O₁₄(M+Na): 646. Found: 646. This material was used directly in the nextstep.

Compound 18

To a solution of 421 mg of partially purified compound 17 (0.68 mmoltheoretical) and 282 μL (2.02 mmol) of Et₃N in 4 mL of CH₂Cl₂ at 0° C.was added a solution of 446 mg (2.02 mmol) of mono-CBZ-piperazine in 4mL of CH₂Cl₂. The mixture was stirred at room temperature for 1.5 hours,cooled back to 0° C., acidified with 1 N HCl, and partitioned between 50mL of 1 N HCl and 3×50 mL of CH₂Cl₂. The combined CH₂Cl₂ layers werewashed with saturated NaHCO₃ solution, dried (MgSO₄), filtered, andconcentrated to give 500 mg of viscous residue. Purification by silicagel chromatography (CH₂Cl₂/MeOH) gave 265 mg (50%) of compound 18 as aviscous oil: ¹H NMR (CDCl₃) δ 1.45 (s, 9H), 3.40-3.70 (m, 28H), 4.28 (t,4H), 5.16 (s, 4H), 7.37 (brd s, 10H); MS (ESI) calculated forC₃₉H₅₅NaN₅₅NaN₅O₁₂ (M+Na): 808. Found: 809.

Compound 19

Compound 18 (77 mg, 0.098 mmol) was dissolved in 1.5 mL of CH₂Cl₂ and1.5 mL of TFA was added. The mixture was stirred for 6 hours andconcentrated. The residue was dissolved in 10 mL of CH₂Cl₂ and theresulting solution was stirred:at 0° C. while 15 mL of saturated NaHCO₃solution was added. The aqueous layer was extracted with 4×10 mL ofCH₂Cl₂, and the combined organic layers were dried (MgSO₄), filtered,and concentrated to give 62 mg (92%) of free amine, compound 18a: ¹H NMR(CDCl₃) δ 2.90 (t, 4H), 3.46 (brd s, 16H), 3.66 (m, 8H), 4.25 (t, 4H),5.16 (s, 4H), 7.36 (brd s, 10H). To a solution of compound 17 in CH₂Cl₂is added the free amine, compound 18a (3 eq.) and pyridine. The mixtureis stirred at room temperature until the reaction appears done by TLC.The product is isolated by extractive workup and purification by silicagel chromatography to give compound 19.

Compound 20

Compound 19 is dissolved in 1/1 CH₂Cl₂/TFA, and stirred at roomtemperature for 1 hour. The mixture is concentrated under vacuum toprovide an amine intermediate, compound 19a. Two equivalents of theintermediate amine is reacted with triethyleneglycol bis-chloroformatein CH₂Cl₂ and pyridine. The product is isolated by extractive workup andpurification by silica gel chromatography to give compound 20.

Compound 20a

In a process similar to that described above for compound 15, compound20 is treated with 30% HBr/AcOH for 30 min. The resulting HBr salt isprecipitated with ether. The solids are collected by centrifugation andwashed with ether. The resulting HBr salt is dried in the desiccatorovernight and dissolved in H₂O. The mixture was stirred at 0° C. andsodium bicarbonate added. A solution of bromoacetic anhydride in dioxaneis added, and the mixture stirred at 0° C. for 15-20 min. To the mixtureis added H₂O, and the mixture is slowly acidified with 1 M H₂SO₄ to a pHof 4. The aqueous layer is extracted with EtOAc which was discarded. Theaqueous layer is then extracted with 8/2 CH₂Cl₂/MeOH. The combinedorganic layers are dried (MgSO₄), filtered and concentrated to givecompound 20a.

Example 4

Synthesis of Tetramer of DEGA/TEG Using Core Propagation Approach

A chemical scheme for the preparation of an tetramer of DEGA/TEG isshown in FIG. 13. The bis-diethyleneglycolamine (compound 7) was reactedtriethyleneglycol bis chloroformate (from which the “core” is derived)to yield the tetrahydroxy compound, compound 21. Compound 21 was thenreacted with para-nitrophenylchloroformate to yield thetetrapara-nitrophenylcarbonate compound (compound L2). Thepara-nitrophenylcarbonate (PNP) group was then converted to a carbamategroup by reaction with mono-CBZ-protected piperazine, yielding compound23. The terminal CBZ-protected amino groups were then converted to thehydrobromide salt of amino group, and further reacted with bromoaceticanhydride to yield reactive bromoacetyl groups at each of the termini incompound 23a.

Compound 21

To a solution of 1.94 g (10.0 mmol) of compound 7 and 1.75 ML (1.30 g,10.0 mmol) of Et₃N at 0° C. a solution of 980 μL (1.31 g, 4.78 mmol) oftriethyleneglycol bis-chloroformate in 35 mL of CH₂Cl₂. The mixture wasstirred for 3 hours at room temperature and concentrated to give 4.84 gof crude compound 21 which was used as is in the next step: ¹H NMR(CDCl₃) δ 3.10 (m, 4H), 3.45-3.78 (m, 32H), 4.24 (m, 4H).

Compound 22

Pyridine (9.3 mL, 114.7 mmol) was added to a stirred solution of 4.84 gof crude compound 21 (4.78 mmol theoretical) and 7.71 g (38.24 mmol) of4-nitrophenylchloroformate at 0° C., and the mixture was stirred for 4hours at room temperature. The mixture was cooled to 0° C. and acidifiedwith 1 N HCl. The mixture was partitioned between 250 mL of 1 N HCl and2×250 mL of CH₂Cl₂. The organic layers were combined, dried (MgSO₄),filtered and concentrated to give 9.81 g of crude product. Purificationby silica gel chromatography (CH₂Cl₂/MeOH) gave 4.40 g (74%) of compound22 as a sticky viscous oil: ¹H NMR (CDCl₃) δ 3.56 (m, 8H), 3.61-3.72 (m,16H), 3.76 (m, 8H), 4.23 (t, 4H), 4.44 (t, 8H), 7.40 (d, 8H), 8.28, (d,8H); HRMS (FAB) calculated for C₅₂H₆₀CsN₆O₃₀ (M+Cs): 1381.2408. Found:1381.2476.

Compound 23

A solution of 106 mg (0.48 mmol) of mono-CBZ-piperazine in 0.5 mL ofCH₂Cl₂ was added to a stirred solution of 100 mg (0.08mmol) of compound22 and 67 μL (0.48 mmol) of Et₃N at 0° C. The mixture was stirred for 18hours at room temperature, cooled to 0° C., and acidified with 1 N HCl.The mixture was partitioned between 5 mL of 1 N HCl and 3×5 mL ofCH₂Cl₂. The organic layers were combined, washed with saturated,NaHCO₃solution, dried (MgSO₄), filtered, and concentrated to give 1 19 mg ofyellow oil. Purification by silica gel :chromatography (CH₂Cl₂/MeOH)gave 81 mg (64%) of compound 23 as a viscous oil: ¹H NMR (CDCl₃) δ 63.48 (brd s, 40H), 3.52-3.74 (m, 24H), 4.24 (t, 12H), 5.14 (s, 8H), 7.35(brd s, 20H); HRMS (FAB) calculated for C₇₆H₁₀₄CsN₁₀O₂₆ (M+Cs):1705.6178. Found: 1705.6269.

Compound 23a

In a process similar to that described above for compound 15, compound23 is treated with 30% HBr/AcOH for 30 min. The resulting HBr salt isprecipitated with ether. The solids are collected by centrifugation andwashed with ether. The resulting HBr salt is dried in the desiccatorovernight and dissolved in H₂O. The mixture was stirred at 0° C. andsodium bicarbonate added. A solution of bromoacetic anhydride in dioxaneis added, and the mixture stirred at 0° C. for 15-20 min. To the mixtureis added H₂O, and the mixture is slowly acidified with 1 M H₂SO₄ to a pHof 4. The aqueous layer is extracted with EtOAc which was discarded. Theaqueous layer is then extracted with 8/2 CH₂Cl₂/MeOH. The combinedorganic layers are dried (MgSO₄), filtered and concentrated to givecompound 23a.

Example 5

Synthesis of Tetramer of DEGA/PTH Using Core Propagation Approach

A chemical scheme for the preparation of an tetramer of DEGA/PTH isshown in FIG. 14A. The bis-diethyleneglycolamine (compound 7) wasreacted with terephthaloyl chloride (from which the “core” is derived)to yield the tetrahydroxy compound, compound 24. Compound 24 was thenreacted with para-nitrophenylchloroformate to yield the tetra.para-nitrophenylcarbonate compound (compound 25). Thepara-nitrophenrylcarbonate (PNP) group was then converted to a carbamategroup by reaction with mono-CBZ-protected piperazine, yielding compound26. The terminal CBZ-protected amino groups were then converted to thehydrobromide salt of amino group, and further reacted with bromoaceticanhydride to yield reactive bromoacetyl groups at each of the termini incompound 26a.

Compound 24

A solution of 300 mg (1.48 mmol) of terephthaloyl chloride in 9 mL ofCH₂Cl₂ was slowly added to a 0° C. solution of 600 mg (3.10 mmol) of 7and 540 μL (3.10 mmol) of diisopropylethylamine in 12 mL of CH₂Cl₂, andthe mixture was stirred under nitrogen atmosphere for 3 hours at roomtemperature. The mixture was concentrated under vacuum to give 1.53 g ofa crude mixture which contained 24.

Compound 25

The 1.53 g of crude 24 and 2.38 g (1 1.81 mmol) of4-nitrophenylchloroformate were dissolved in 30 mL of pyridine, and theresulting solution was stirred at 0C while 1.91 mL (23.62 mmol) ofpyridine was added. The mixture was stirred at room temperature for 4hours, cooled to 0° C., and acidified with 1 N HCl. The mixture waspartitioned between 75 mL of 1 N HCl and 2×75 mL of CH₂Cl₂. The organiclayers were combined, washed with saturated NaHCO₃ solution, dried(MgSO₄), filtered, and concentrated to give 2.75 g of an oil.Purification by silica gel chromatography (CH₂Cl₂/MeOH) gave 1.33 g(76%) of 25 as a viscous oil: ¹H NMR (CDCl₃) δ 3.52 (brd s, 8H), 3.65(brd s, 4H), 3.81 (brd s, 12H), 4.41 (m, 8H), 7.38 (m, 8H), 7.47 (s,4H), 8.27 (m, 8H); HRMS (FAB) calculated for C₅₂H₅₃N₆O₂₆ (M+H):1177.3010. Found: 1177.3062.

Compound 26

A solution of 113 mg (0.51 mmol3.of mono-CBZ-piperazine in 0.5 mL ofCH₂Cl₂ was added to a stirred solution of 100 mg (0.085 mmol) ofcompound 25 and 71 μL (0.51 mmol) of Et₃N at 0° C. The mixture wasstirred for 18 hours at room temperature, cooled to 0° C., and acidifiedwith 1 N HCl. The mixture was partitioned between 5 mL of 1 N HCl and3×5 mL of CH₂Cl₂. The organic layers were combined, washed withsaturated NaHCO₃ solution, dried (MgSO₄), filtered, and concentrated togive 125 mg of yellow oil. Purification by silica gel chromatography(CH₂Cl₂/MeOH) gave 59 mg (46%) of 26 as a viscous oil: ¹H NMR (CDCl₃) δ3.45 (brd s, 40H), 3.55 (m, 4H), 3.72 (m, 4H), 3.78 (s, 8H), 4.24 (m,8H), 5.13 (s, 8H), 7.34 (brd s, 20H), 7.42 (s, 4H); HRMS (FAB)calculated for C₇₆H₉₆CsN₁₀O₂₂ (M+Cs): 1633.5755. Found: 1633.5846.

Compound 26a

In a process similar to that described above for compound 15, compound26 is treated with 30% HBr/AcOH for 30 min. The resulting HBr salt isprecipitated with ether. The solids are collected by centrifugation andwashed with ether. The resulting HBr salt is dried in the desiccatorovernight and dissolved in H₂O. The mixture was stirred at 0° C. andsodium bicarbonate added. A solution of bromoacetic-anhydride: indioxane is added, and the mixture stirred at 0° C. for 15-20 min. To themixture is added H₂O; and the mixture is slowly acidified with 1 M H₂SO₄to a pH of 4. The aqueous layer is extracted with EtOAc which wasdiscarded. The aqueous layer is then extracted with 8/2 CH₂Cl₂/MeOH. Thecombined organic layers are dried (MgSO₄), filtered and concentrated togive compound 26a.

Example 6

Synthesis of Octamer of DEGA/PTH Using Core Propagation Approach

A chemical scheme for the preparation of an octamer of DEGA/PTH is shownin FIG. 14B. The bis-diethyleneglycolamine (compound 7) was reacted withthe tetra para-nitrophenylcarbonate compound (compound 25) to yield theoctahydroxy compound, compound 27a. Compound 27a was then: reacted withpara-nitrophenylchloroformate to yield the octapara-nitrophenylcarbonate compound (compound 27). Thepara-nitrophenylcarbonate (PNP) group was then converted to a carbamategroup by reaction with mono-CBZ-protected piperazine, yielding compound28. The terminal CBZ-protected amino groups were then converted to thehydrobromide salt of amino group, and further reacted with bromoaceticanhydride to yield reactive bromoacetyl groups at each of the termini incompound 28a.

Compound 27a

A solution of 98 mg (0.51 mmol) of compound 7 in 0.5 mL of CH₂Cl₂ wasadded to a stirred solution of 100 mg (0.085 mmol) of compound 25 and 71μL (0.51 mmol) of Et₃N at 0° C., and the mixture was stirred for 18hours. The mixture was concentrated to give 250 mg of crude product,compound 27a.

Compound 27

Crude compound 27a (250 mg) was dissolved in 4 mL of CH₂Cl₂. The mixturewas cooled to 0° C., and 275 mg (1.36 mmol) of4-nitrophenylchloroformate was added followed by 220 μL (2.74 mmol) ofpyridine. The mixture was stirred at room temperature for 7 hours,cooled to 0° C., and acidified with 1 N HCl. The mixture was partitionedbetween 5 mL of 1 N HCl and 2×15 mL of CH₂Cl₂. The organic layers werecombined, washed with saturated NaHCO₃ solution, dried (MgSO₄),filtered, and concentrated to give 393 mg of an oil. Purification bysilica gel chromatography (CH₂Cl₂/MeOH) gave 123 mg (53%) of compound 27as a viscous oil: ¹H NMR (CDCl₃) δ 3.63-3.86 (m, 72H), 4.46 (t, 24H),7.26 (s, 4H), 7.35 (m, 16H), 8.24 (m, 16H).

Compound 28

A solution of 6 eq. of mono-CBZ-piperazine in CH₂Cl₂ is added to astirred solution of compound 27 and 6 eq. of Et₃N at 0° C. The mixtureis stirred for 18 hours at room temperature, cooled to 0° C., andacidified with 1 N HCl. The mixture is partitioned between 1 N HCl andCH₂Cl₂. The organic layers are combined, washed with saturated NaHCO₃solution, dried (MgSO₄), filtered, and concentrated to give crudeproduct which can be purified by silica gel chromatography to givecompound 28.

Compound 28a

In a process similar to that described above for compound 15, compound28 is treated with 30% HBr/AcOH for 30 min. The resulting HBr salt isprecipitated with ether. The solids are collected by centrifugation andwashed with ether. The resulting HBr salt is dried in the desiccatorovernight and dissolved in H₂O. The mixture was stirred at 0° C. andsodium bicarbonate added. A solution of bromoacetic anhydride in dioxaneis added, and the mixture stirred at 0° C. for 15-20 min. To the mixtureis added H₂O, and the mixture is slowly acidified with 1 M H₂SO₄ to a pHof 4. The aqueous layer is extracted with EtOAc which was discarded. Theaqueous layer is then extracted with 8/2 CH₂Cl₂/MeOH. The combinedorganic layers are dried (MgSO₄), filtered and concentrated to givecompound 28a.

Example 7

Synthesis of Tetramer of HEGA/TEG Using Core Propagation Approach

A chemical scheme for the preparation of an tetramer of HEGA/TEG isshown in FIG. 15. The bis-hexaethyleneglycolamine (compound 7) wasreacted triethyleneglycol bis chloroformate (from which the “core” isderived) to yield the tetrahydroxy compound, compound 29. Compound 29was then reacted with para-nitrophenylchloroformate to yield the tetrapara-nitrophenylcarbonate compound (compound 30). Thepara-nitrophenylcarbonate (PNP) group was then converted to a carbamategroup by reaction with mono-CBZ-protected piperazine, yielding compound31. The terminal CBZ-protected amino groups were then converted to thehydrobromide salt of amino group, and further reacted with bromoaceticanhydride to yield reactive bromoacetyl groups at each of the termini incompound 31a.

Compound 29

To a solution of 2.1 eq. of compound 4 and 2.1 eq. of Et₃N at 0° C. isadded a solution of 1 eq. of triethyleneglycol bis-chloroformate inCH₂Cl₂. The mixture is stirred at room temperature until complete by TLCand concentrated to give crude compound 29 which is used as is in thenext step.

Compound 30

Pyridine (12 eq.) is added to a stirred solution of crude compound 29and 6 eq. of 4-nitrophenylchloroformate at 0° C., and the mixture isstirred at room temperature until the reaction is complete as evidencedby TLC. The mixture is cooled to 0° C., and acidified with 1 N HCl. Themixture is partitioned between 1 N HCl and CH₂Cl₂. The organic layersare combined, dried (MgSO₄), filtered and concentrated to give crudeproduct. Compound 30 is purified by silica gel chromatography.

Compound 31

A solution of 6 eq. of mono-CBZ-piperazine in CH₂Cl₂ is added to astirred solution of compound 30 and 6 eq. of Et₃N at 0° C. The mixtureis stirred for at room temperature until the reaction is complete asevidenced by TLC, cooled to 0° C., and acidified with 1 N HCl. Themixture is partitioned between 1 N HCl and CH₂Cl₂. The organic layersare combined, washed with saturated NaHCO₃ solution, dried (MgSO₄),filtered, and concentrated to give crude compound 31 which is purifiedby silica gel chromatography.

Compound 31a

In a process similar to that described above for compound 15, compound31 is treated with 30% HBr/AcOH for 30 min. The resulting HBr salt isprecipitated with ether. The solids are collected by centrifugation andwashed with ether. The resulting HBr salt is dried in the desiccatorovernight and dissolved in H₂O. The mixture was stirred at 0° C. andsodium bicarbonate added. A solution of bromoacetic anhydride in dioxaneis added, and the mixture stirred at 0° C. for 15-20 min. To the mixtureis added H₂O, and the mixture is slowly acidified with 1 M H₂SO₄ to a pHof 4. The aqueous layer is extracted with EtOAc which was discarded. Theaqueous layer is then extracted with 8/2 CH₂Cl₂/MeOH. The combinedorganic layers are dried (MgSO₄), filtered and concentrated to givecompound 31a.

Example 8

Synthesis of Tetramer of DEA/PTH Using Core Propagation Approach

A chemical scheme for the preparation of an tetramer of DEGA/PTH isshown in FIG. 16A. Diethanolamine was reacted with terephthaloylchloride (from which the “core” is derived) to yield the tetrahydroxycompound, compound 32. Compound 32 was then reacted withpara-nitrophenylchloroformate to yield the tetrapara-nitrophenylcarbonate compound (compound 33). Thepara-nitrophenylcarbonate (PNP) group was then converted to a carbamategroup by reaction with mono-CBZ-protected piperazine, yielding compound34. The terminal CBZ-protected amino groups were then converted to thehydrobromide salt of amino group, and further reacted with bromoaceticanhydride to yield reactive bromoacetyl groups at each of the termini incompound 34a.

Compound 32

A solution of 2.0 g (9.85 mmol) of terephthaloyl chloride in 25 mL ofTHF was added slowly to a 0° C. solution of 2.17 g (20.7 mmol) ofdiethanolamine and 3.6 mL (20.7 mmol) of diisopropylethylamine in 50 mLof THF. The mixture was stirred at room temperature for 3 hours andconcentrated under vacuum to give 6.7 g of crude compound 32. A smallamount was purified for characterization purposes by preparative HPLC(1″ C18 column, gradient 0-15% B, A=0.1% TFA/H₂O and B=0.1% TFA/CH₃CN):¹H NMR (D₂O) δ 3.52 (m, 4H), 3.59 (m, 4H), 3.72 (m, 4H), 3.89 (m, 4H),7.51 (s, 4H); MS (ESI) calculated for C₁₆H₂₅N₂O₆ (M+H): 341. Found: 341.

Compound 33

Pyridine (11.4 mL, 141.2 mmol) was slowly added to a stirred solution of6.01 g (8.8 mmol theoretical) of crude compound 32 and 14.2 g (70.6mmol) of 4-nitrophenylchloroformate in 88 mL of THF. The mixture wasstirred at room temperature for 18 hours and acidified with 1 N HCl. Themixture was partitioned between 300 mL of 1 N HCl and 2×300 mL ofCH₂Cl₂. The combined organic layers were washed with saturated NaHCO₃solution, dried (MgSO₄), filtered, and concentrated to give 14.0 g ofsticky orange oil. Purification by silica gel chromatography(CH₂Cl₂/MeOH) provided 3.34 g (38%) of compound 33 as a crystallinesolid: mp 77-85° C.; ¹H NMR (CDCl₃) δ 3.76 (brd, 4H), 4.01 (brd, 4H),4.38 (brd, 4H), 4.64 (brd, 4H), 7.36 (brd, 8H), 7.53 (s, 4H), 8.28 (m,8.28 (m, 8H); ¹³C NMR (CDCl₃) δ 45.0, 48.5, 65.9, 66.6, 121.8, 125.0,125.4, 126.1, 127.2, 137.1, 145.6, 152.4, 155.2, 171.9; HRMS (FAB)calculated for C₄₄H₃₆CsN₆O₂₂ (M+Cs): 1133.0937. Found: 1133.0988.

Compound 34

A solution of 132 mg (0.6 mmol) of mono-CBZ-piperazine in 0.5 mL ofCH₂Cl₂ was added to a 0° C. solution of 100 mg (0.1 mmol) of compound 33and 84 μL (0.6 mmol) of Et₃N in 0.5 mL of CH₂Cl₂. The reaction mixturewas stirred for 18 hours at room temperature, cooled to 0° C., andacidified with 1 N HCl. The mixture was partitioned between 5 mL of 1 NHCl and 3×5 mL of CH₂Cl₂. The combined organic layers were washed withsaturated NaHCO₃ solution, dried (MgSO₄), filtered, and concentrated togive 123 mg of white solid. Purification by silica gel chromatography(CH₂Cl₂/MeOH) provided 86 mg (65%) of compound 34 as a crystallinesolid: mp 64-67° C.; 1H NMR (CDCl₃) δ 3.35-3.63 (m, 3.6H), 3.86 (brd,4H), 4.13 (brd, 4H), 4.41 (brd, 4H), 5.14 (s, 8H), 7.38 (brd s, 20H),7.43 (s, 4H); ¹³C NMR(CDCl₃) δ 43.5, 45.1, 48.4, 62.6, 67.4, 126.9,128.0, 128.2, 128.5, 136.3, 137.4, 155.1, 171.4; HRMS (FAB) calculatedfor C₆₈H₈₀CsN₁₀O₁₈ (M+Cs): 1457.4706. Found: 1457.4781.

Compound 34a

In a process similar to that described above for compound 15, compound34 is treated with 30% HBr/AcOH for 30 min. The resulting HBr salt isprecipitated with ether. The solids are collected by centrifugation andwashed with ether. The resulting HBr salt is dried in the desiccatorovernight and dissolved in H₂O. The mixture was stirred at 0° C. andsodium bicarbonate added. A solution of bromoacetic anhydride in dioxaneis added, and the mixture stirred at 0° C. for 15-20 min. To the mixtureis added H₂O, and the mixture is slowly acidified with 1 M H₂SO₄ to a pHof 4. The aqueous layer is extracted with EtOAc which was discarded. Theaqueous layer is then extracted with 8/2 CH₂Cl₂/MeOH. The combinedorganic layers are dried (MgSO₄), filtered and concentrated to givecompound 34a.

Example 9

Synthesis of Octamer of DEA/PTH Using Core Propagation Approach

A chemical scheme for the preparation of an octamer of DEA/PTH is shownin FIG. 16B. Diethanolamine was reacted with thetetrapara-nitrophenylcarbonate compound (compound 33) to yield theoctahydroxy compound, compound 35a. Compound 35a was then reacted withpara-nitrophenylchloroformate to yield the octapara-nitrophenylcarbonatecompound (compound 35). The para-nitrophenylcarbonate (PNP) group wasthen converted to a carbamate group by reaction with mono-CBZ-protectedpiperazine, yielding compound 36. The terminal CBZ-protected aminogroups were then converted to the hydrobromide salt of amino group, andfurther reacted with bromoacetic anhydride to yield reactive bromoacetylgroups at each of the termini in compound 36a.

Compound 35a

A solution of 100 mg (0.1 mmol) of compound 33 in 450 μL of pyridine wasslowly added to a 0° C. solution of 63 mg (0.6 mmol) of diethanolaminein 150 μL of pyridine. The mixture was stirred for 3 hours at roomtemperature and cooled back to 0° C., to yield crude compound 35a, whichwas used in the next step.

Compound 35

A solution of 443 mg (2.2 mmol) of 4-nitrophenylchloroformate was addedto the reaction mixture above, and the mixture was stirred for 18 hoursat room temperature. The mixture was then cooled to 0° C., and acidifiedwith 1 N HCl, and partitioned between 15 mL of 1 N HCl and 2×15 mL ofCH₂Cl₂. The combined organic layers were dried (MgSO₄), filtered, andconcentrated to give 462 mg of white sticky solid. Purification bysilica gel chromatography (CH₂Cl₂/MeOH) provided 141 mg (65%) ofcompound 35 as a crystalline solid: mp 75-80° C; ¹H NMR (CDCl₃) δ3.52-3.81 (m, 20H), 3.89 (m, 4H), 4.12 (m, 4H), 4.36-4.59 (m, 20H), 7.42(m, 20H), 8.30 (m, 16H).

Compound 36

A solution of 61 mg (0.276 mmol) of mono-CBZ-piperazine in 200 [L ofCH₂Cl₂ was added to a 0° C. solution of 50 mg (0.023 mmol) of compound35 and 39 μL (0.276 mmol) of Et₃N in 200 μL of CH₂Cl₂. The reactionmixture was stirred for 7 hours at room temperature, cooled to 0° C.,and acidified with 1 N HCl. The mixture was partitioned between 10 mL of1 N HCl and 3×10 mL of CH₂Cl₂. The combined organic layers were dried(MgSO₄), filtered, and concentrated to give 85 mg of yellow solid.Purification by silica gel chromatography (CH₂Cl2/MeOH) provided 33 mg(51%) of compound 36 as a sticky low melting solid: ¹H NMR (CDCl₃) δ3.37-3.72 (m, 84H), 3.84 (m, 4H), 4.03-4.29 (m, 20H), 4.35 (m, 4H), 5.14(s, 16H), 7.35 (brd s, 40H), 7.44 (s, 4H); MS (MALDI) calculated for C₁₄H₁₇₂NaN₂₂O₄₂ (M+Na): 2856. Found: 2857.

Compound 36a

In a process similar to that described above for compound 15, compound36 is treated with 30% HBr/AcOH for 30 min. The resulting HBr salt isprecipitated with ether. The solids are collected by centrifugation andwashed with ether. The resulting HBr salt is dried in the desiccatorovernight and dissolved in H₂0. The mixture was stirred at 0° C. andsodium bicarbonate added. A solution of bromoacetic anhydride in dioxaneis added, and the mixture stirred at 0° C. for 15-20 min. To the mixtureis added H₂O, and the mixture is slowly acidified with 1 M H₂SO₄ to a pHof 4. The aqueous layer is extracted with EtOAc which was discarded. Theaqueous layer is then extracted with 8/2 CH₂Cl₂/MeOH. The combinedorganic layers are dried (MgSO₄), filtered and concentrated to givecompound 36a.

Example 10

Synthesis of Tetramer of DEA/DEG Using Core Propagation Approach

A chemical scheme for the preparation of an tetramer of DEA/DEG is shownin FIGS. 17A and 17B. Diethyleneglycol (from which the“core” is derived)was reacted with para-nitrophenylchloroformate to yield the dipara-nitrophenylcarbonate compound. (compound 37). Compound 37 was thenreacted with diethanolamine to form the tetrahydroxy compound, compound38. Compound 38 was then reacted with para-nitrophenylchloroformate toyield the tetrapara-nitrophenylcarbonate compound (compound 39). Thepara-nitrophenylcarbonate (PNP) group was then converted to a carbamategroup by reaction with mono-CBZ-protected piperazine, yielding compound40. The terminal CBZ-protected amino groups were then converted to thehydrobromide salt of amino group (compound 41), and further reacted withbromoacetic anhydride to yield reactive bromoacetyl groups at each ofthe termini in compound 42.

Compound 37 Diethyleneglycol bis-4-nitrophenylcarbonate

Pyridine (30.5 mL, 377 mmol) was slowly added to a 0° C. solution of 5.0g (47.11 mmol) of diethylene glycol and 23.74 g (118 mmol) of4-nitrophenylchloroformate in 500 mL of THF. The cooling bath wasremoved, and the mixture was stirred for 18 hours at room temperature.The mixture was cooled back to 0° C., acidified with 6 N HCl, andpartitioned between 400 mL of 1 N HCl and 2×400 mL of CH₂Cl₂. Thecombined organic layers were dried (MgSO₄), filtered, and concentratedto give 24.3 g of a white solid. Crystallization from hexanes/EtOAc gave16.0 g (78%) of compound 37 as a white powder: mp 110° C.; ¹H NMR(CDCl₃) δ 3.89 (t, 4H), 4.50 (t, 4H), 7.40 (d, 4H), 8.26 (d, 4H).

Compound 38

A solution of 2.5 g (5.73 mmol) of compound 37 in 17 mL of pyridine wasadded to a 0° C. solution of 1.8 g (17.2 mmol) of diethanolamine in 3 mLof pyridine. The cooling bath was removed, and the mixture was stirredfor 5 hours at room temperature to yield compound 38, which was notisolated but was used as is in the next step.

Compound 39

The mixture from the previous step was cooled back to 0° C., 40 mL ofCH₂Cl₂ was added followed by a solution of 11.55 g (57.3 mmol) of4-nitrophenylchloroformate in 60 mL of CH₂Cl₂, and the mixture wasstirred for 20 hours at room temperature. The mixture was cooled back to0° C., acidified with 1 N HCl, and partitioned between 300 mL of 1 N HCland 2×200 mL of CH₂Cl₂. The combined organic layers were dried (MgSO₄),filtered, and concentrated to give 13.6 g of yellow solid. Purificationby silica gel chromatography (CH₂Cl₂/MeOH and EtOAc/hexanes) provided4.91 g (83%) of compound 39 as a sticky amorphous solid: ¹H NMR (CDCl₃)δ 3.72 (m, 12H), 4.31 (t, 4H), 4.48 (m, 8H), 7.40 (m, 8H), 8.29 (m, 8H).

Compound 40

A solution of 128 mg (0.58 mmol) of mono-CBZ-piperazine in 1.0 mL ofCH₂Cl₂ was added to a 0° C. solution of 100 mg (0.10 mmol) of compound39 and 82 μL (0.58 mmol) of Et₃N in 1.0 mL of CH₂Cl₂. The reactionmixture was stirred for 18 hours at room temperature, cooled to 0° C.,and acidified with 1 N HCl. The mixture was partitioned between 10 mL of1 N HCl and 3×10 mL of CH₂Cl₂. The combined organic layers were washedwith saturated NaHCO₃ solution, dried (MgSO₄), filtered, andconcentrated to give 162 mg of yellow oil.

Purification by silica gel chromatography (EtOAc/hexanes followed byCH₂Cl₂/MeOH) provided 125 mg (95%) of compound 40 as a glassy amorphoussolid: ¹H NMR (CDCl₃) δ 3.36-3.59 (m, 40H), 3.68 (t, 4H), 4.22 (m, 12H),5.16, (s, 8H), 7.36 (brd s, 20H); MS (ESI) calculated for C₆₆H₂₄NaN₁₀O₂₁(M+Na): 1376. Found: 1376.

Compound 41

Compound 40 (150 mg, 0.1 1 mmol) was dissolved in 3 mL of 30% HBr/HOAc.The mixture was stirred for 30 minutes at room temperature, and the HBrsalt was precipitated by addition of Et₂O. The precipitate, compound 41,was washed twice with Et₂O and dried under vacuum.

Compound 42

The precipitate, compound 41, was dissolved in 20 mL of H₂O. The mixturewas brought to pH 12 by addition of 10 N NaOH and extracted with six 20mL portions of 4/1 CH₂Cl₂/MeOH. The combined organic layers were dried(MgSO₄), filtered, and concentrated to provide the free amine. The freeamine was dissolved in 8 mL of CH₃CN, 1.5 mL of MeOH was added toimprove solubility, and the solution was stirred at 0° C. while asolution of 114 mg (0.66 mmol) of chloroacetic anhydride in 2 mL ofCH₃CN was added. The mixture was stirred at room temperature for 2.5hours and concentrated to give 107 mg of an oil. Purification bypreparative HPLC (1″ C18 column, gradient 25-45% B, A=0.1% TFA/H₂O andB=0.1% TFA/CH₃CN) provided 44 mg of compound 42 as a viscous oil: ¹H NMR(CDCl₃) δ 3.41-3.80 (m, 44H), 4.12 (s, 8H), 4.24 (m, 12H); MS (ESI)calculated for C₄₂H₆₅Cl₄N₁₀O₁₇(M+H): 1121. Found: 1121.

Example 11

Synthesis of Octamer of DEA/DEG Using Core Propagation Approach

A chemical scheme for the preparation of an octamer of DEA/DEG is shownin FIGS. 17C and 17D. The tetrapara-nitrophenylcarbonate compound,compound 39, was reacted with diethanolamine to form the octahydroxycompound, compound 43a. Compound 43a was then reacted withpara-nitrophenylchloroformate to yield the octapara-nitrophenylcarbonatecompound (compound 43). The para-nitrophenylcarbonate (PNP) group wasthen converted to a carbamate group by reaction with mono-CBZ-protectedpiperazine, yielding compound 44. The terminal CBZ-protected aminogroups were then converted to the hydrobromide salt of amino group(compound 4a), and further reacted with bromoacetic anhydride to yieldreactive bromoacetyl groups at each of the termini in-compound 45.

Compound 43a

A solution of 100 mg (0.097 mmol) of compound 39 in 450 μL of pyridinewas added to a 0° C. solution of 61 mg (0.583 mmol) of diethanolamine in150 ZL of pyridine. The cooling bath was removed, and the mixture wasstirred for 20 hours at room temperature, to yield the crude compound43a, which was used as is in the next step.

Compound 43

The mixture from the previous step was cooled back to 0° C., and asolution of 431 mg (2.138 mmol) of 4-nitrophenylchloroformate in 4 m]Lof THF followed by 2 mL of CH₂Cl₂ to improve solubility. The mixture wasstirred for 18 hours at room temperature, cooled back to 0° C.,acidified with 1 N HCl, and partitioned between 20 mL of 1 N HCl and2×20 mL of CH₂Cl₂. The combined organic layers were dried (MgSO₄),filtered, and concentrated to give 505 mg of yellow solid. Purificationby silica gel chromatography (EtOAc/hexanes) provided 146 mg (68%) ofcompound 43 as a crystalline solid: mp 67-69° C.; ¹H NMR (CDCl₃) δ3.50-3.80 (m, 28H), 4.22 (m, 12H), 4.43 (m, 16H), 7.40 (m, 16H), 8.30(m, 16H).

Compound 44

A solution of 476 mg (2.16 mmol) of mono-CBZ-piperazine in 2.0 mL ofCH₂Cl₂ was added to a 0° C. solution of 400 mg (0.18 mmol) of compound43 and 300 ∥I (2.16 mmol) of Et₃N in 2.0 mL of CH₂Cl₂. The reactionmixture was stirred for 18 hours at room temperature, cooled to 0° C.,and acidified with 1 N HCl. The mixture was partitioned between 40 mL of1 N HCl and 3×40 mL of CH₂Cl₂. The combined organic layers were washedwith 3×40 mL of saturated NaHCO₃ solution, dried (MgSO₄), filtered, andconcentrated to give 483 mg (97%) of compound 44 as a sticky white solidwhich was pure enough for use inthe next step: ¹H NMR (CDCl₃) δ3.36-3.60 (m, 88H), 3.66 (t, 4H), 4.21 (brd, 28H), 5.15, (s, 16), 7.36(brd s, 40H).

Compound 44a

Compound 44 (150 mg, 0.054 mmol) was dissolved in 2 mL of 30% HBr/HOAc.The mixture was stirred for 30 minutes at room temperature, and the HBrsalt was precipitated by addition of Et₂O. The precipitate was washedtwice with Et₂O, and dried under vacuum.

Compound 45

The precipitate, compound 44a, was dissolved in 20 mL of H₂O. Themixture was brought to pH 12 by addition of 10 N NaOH and extracted withsix 20 mL portions of 4/1 CH₂Cl₂/MeOH. The combined organic layers weredried (MgSO₄), filtered, and concentrated to provide the free amine. Thefree amine was dissolved in 4 mL of CH₃CN, 0.5 mL of MeOH was added toimprove solubility, and the solution was stirred at 0° C. while asolution of 112 mg (0.65 mmol) of chloroacetic anhydride in 1 mL ofCH₃CN was added. The mixture was stirred at room temperature for 2 hoursand concentrated to give 111 mg of an oil. Purification by preparativeHPLC (1″ C18 column, gradient 35-55% B, A=0.1% TFA/H₂O and B=0.1%TFA/CH₃CN) provided 20 mg (15%) of compound 45 as a viscous oil: ¹H NMR(CDCl₃) δ 3.40-3.78 (m, 92H), 4.15 (S, 16H), 4.28, (m, 28H).

Example 12

Synthesis of Tetramer and Octamer of ADP/DEG Using Core PropagationApproach

A chemical scheme for the preparation of an octamer of ADP/DEG is shownin FIGS. 18A and 18B. The di-para-nitrophenylcarbonate derivative ofdiethyleneglycol, compound 37. (from which the “core” is derived) wasreacted with 2-amino-1,3-propanediol to yield the tetrahydroxy compound,compound 46. Compound 46 was reacted withpara-nitrophenylchloroformateto yield the tetrapara-nitrophenylcarbonatecompound (compound 7). Compound 47 was then reacted with2-amino-1,3-propanediol to yield the octahydroxy compound, compound 47a.Compound 47a was then reacted with para-nitrophenylchloroformate toyield the octapara-nitrophenylcarbonate compound (compound 48). Thepara-nitrophenylcarbonate (PNP) group was then converted to a carbamategroup by reaction with mono-CBZ-protected piperazine, yielding compound48a. The terminal CBZ-protected amino groups were then converted to thehydrobromide salt of amino group (compound 48b), and further reactedwith chloroacetic anhydride to yield reactive chloroacetyl groups ateach of the termini in compound 48c.

Compound 46

A solution of compound 37 in pyridine is added to a 0° C. solution of 3eq. of 2-amino-1,3-propanediol in pyridine. The cooling bath is removed,and the mixture is stirred for 5 hours at room temperature. Compound 46can be isolated if desired, however, it is generally more convenient toisolate after forming the 4-nitrophenylcarbonate ester.

Compound 47

The mixture above is cooled back to 0° C., a solution of 10 eq. of4-nitrophenylchloroformate in 60 mL of CH₂Cl₂ is added, and the mixtureis stirred for 20 hours at room temperature. The mixture is cooled backto 0° C., acidified with 1 N HCl, and is partitioned between 1 N HCl andCH₂Cl₂. The combined organic layers are dried (MgSO₄), filtered, andconcentrated. Purification by silica gel chromatography providescompound 47.

Compound 47a

A solution of compound 47 in pyridine is added to a 0° C. solution of 6eq. of 2-amino-1,3-propanediol in pyridine. The cooling bath is removed,and the mixture is stirred for 20 hours at room temperature to yieldcompound 47a, which is used in the next step.

Compound 48

The mixture above is cooled back to 0° C., and a solution of4-nitrophenylchloroformate is added. The mixture is stirred for 18 hoursat room temperature, cooled back to 0° C., acidified with 1 N HCl, andpartitioned between 1 N HCl and CH₂Cl₂. The combined organic layers aredried (MgSO₄), filtered, and concentrated. Purification by silica gelchromatography provides compound 48.

Compound 48a

In a manner similar to that for compound 44 above, a solution ofmono-CBZ-piperazine in CH₂Cl₂ is added to a 0° C. solution of compound43 and Et₃N in CH₂Cl₂. The reaction mixture was stirred for 18 hours atroom temperature, cooled to 0° C., and acidified with 1 N HCl. Themixture was partitioned between 1 N HCl and CH₂Cl₂. The combined organiclayers are washed with saturated NaHCO₃ solution, dried (MgSO₄),filtered, and concentrated to give compound 48a, for use in the nextstep.

Compound 48b

In a manner similar to that for compound 44a above, compound 48a isdissolved in 30% HBr/HOAc. The rnixture is stirred for 30 minutes atroom temperature, and the HBr salt precipitated by addition of Et₂O. Theprecipitate is washed with Et₂O, and dried under vacuum.

Compound 48c

In a manner similar to that for compound 45above, the precipitate,compound 48b, is dissolved in H₂O. The mixture is brought to pH 12 byaddition of 10 N NaOH and extracted with 4/1 CH₂Cl₂/MeOH. The combinedorganic layers are dried (MgSO₄), filtered, and concentrated to providethe free amine. The free amine is dissolved in CH₃CN, MeOH is added toimprove solubility, and the solution is stirred at 0° C. while asolution of chloroacetic anhydride in CH₃CN is added. The mixture wasstirred at room temperature for 2 hours and concentrated to give crudecompound 48c, which is purified by preparative HPLC.

Example 13

Synthesis of Octamer of DEA/PE Using Core Propagation Approach

A chemical scheme for the preparation of an octamer of DEA/PE is shownin FIG. 19. Pentaerythritol (from which the “core” is derived) wasreacted with para-nitrophenylchloroformate to yield thetetrapara-nitrophenylcarbonate compound (compound 49). Compound 49 wasthen reacted with diethanolamine to form the octahydroxy compound,compound 49a. Compound 49a was then reacted withpara-nitrophenylchloroformate to yield the octapara-nitrophenylcarbonatecompound (compound. 5). The para-nitrophenylcarbonate (PNP) group wasthen converted to a carbamate group by reaction withmono-N-BOC-ethylenediamine, yielding compound 51.

Compound 49 Pentaerythritol tetrakis-4-nitrophenylcarbonate

Pyridine (950 μL, 11.74 mmol) was slowly added to a 0° C. solution of100 mg (0.734 mmol) of pentaerythritol and 1.18 g (5.88 mmol) of4-nitrophenylchloroformate in 10 mL of CH₂Cl₂. The cooling bath wasremoved, and the mixture was stirred for 24 hours at room temperature.The mixture was cooled back to 0° C., acidified with 1 N HCl, andpartitioned between 50 mL of 1 N HCl and 2×50 mL of CH₂Cl₂. The combinedorganic layers were dried (MgSO₄), filtered, and concentrated to give1.123 g of a white solid. Purification by silica gel chromatography(EtOAc/hexanes followed by CH₂Cl₂/MeOH) provided 128 mg (22%) ofcompound 49 as a white crystalline solid: mp 175° C.;: ¹H NMR (CDCl₃) δ4.61 (s, 8H), 7.40, (m, 8H), 8.30 (m, 8H).

Compound 49a

To solution of 120 mg (0.150 mmol) of compound 49 in 1.6 mL of pyridineat 0° C. was added 87 μL (95 mg, 0.90 mmol) of diethanolamine. Thecooling bath was removed, and the mixture was stirred for 7 hours atroom temperature, and cooled back to 0° C., to yield compound 49a, whichwas used as is in the next step.

Compound 50

A solution of 756 mg (3.75 mmol) of 4-nitrophenylchloroformate in 3 mLof CH₂Cl₂ was added the mixture above. The mixture was stirred for 18hours at room temperature, cooled back to 0° C., acidified with 1 N HCl,and partitioned between 20 mL of 1 N HCl and 2×20 mL of CH₂Cl₂. Thecombined organic layers were dried (MgSO₄), filtered, and concentratedto give 819 mg of sticky yellow solid. Purification by silica gelchromatography (EtOAc/hexanes and CH₂Cl₂/MeOH) provided 134 mg (47%) ofcompound 50 as a sticky viscous oil with some impurities: H NMR (CDCl₃)δ 3.69 (m, 16H), 4.31 (s, 8H), 4.41 (m, 16H), 7.39 (m, 16H), 8.25 (m,16H).

Compound 51

A solution of compound 50 is treated with 10 eq. ofmono-N-BOC-ethylenediamine in pyridine and CH₂Cl₂. The mixture isstirred at room temperature until complete as evidenced by TLC, andpartitioned between 1 N HCl and CH₂Cl₂. The combined organic layers isdried (MgSO₄), filtered, and concentrated to give crude product.Purification by silica gel chromatography (EtOAc/hexanes andCH₂Cl₂/MeOH) provides compound 51.

Example 14

Solid Phase Synthesis of Tetramer of DEA/DEG Using Segmental Approach

A chemical scheme for the solid phase synthesis of a tetramer of octamerof DEA/DEG is shown in FIGS. 20A and 20B. Wang resin, having terminalhydroxy groups, was reacted with para-nitrophenylchloroformate to yieldthe para-nitrophenylcarbonate compound (compound 52). Compound 52 wasthen reacted with diethanolamine to form the dihydroxy compound,compound 52a. Compound 52a was then reacted withpara-nitrophenylchloroformate to yield the di para-nitrophenylcarbonatecompound (compound 52b). The para-nitrophenylcarbonate (PNP) group wasthen converted to a carbamate group by reaction with mono-CBZ-protectedpiperazine, yielding compound 53. The CBZ-protected compound was thencleaved from the resin, and reacted with diethyleneglycol bischloroformate (from which the “core” is derived), to yield thetetra-CBZ-protected amino compound, compound 40. The terminalCBZ-protected amino groups may be converted to the hydrobromide salt ofamino group, and further reacted with chloroacetic anhydride to yieldreactive chloroacetyl groups at each of the termini.

Compound 52

Wang resin (25 mg, subst. 0.58 mmol/g, 0.0145 mmol) was washed withCH₂Cl₂. The resin was suspended in 580 μL of CH₂Cl₂, and 15 mg (0.145mmol) of 4-nitrophenylchloroformate was added followed by 97 μL ofpyridine. After gentle agitation of the mixture for 4 hours, the resinwas washed with CH₂Cl₂ and dried, to yield compound 52.

Compound 52a

The resin was then suspended in 410 μL of CH₂Cl₂, and 71 mg (0.673 mmol)of diethanolamine (410 μL of a solution of 82.5 mg ofdiethanolaminedissolved in 493 μL of pyridine). After gentle agitationof the mixture for 16 hours, the resin was washed with CH₂Cl₂ and dried,to yield compound 52a.

Compound 52b

To the resin was added 580 μL of CH₂Cl₂, and to the mixture was added15.2 mg (0.145 mmol) of 4-nitrophenylchloroformate followed by 97 μL ofpyridine. After gentle agitation of the mixture for 4 hours, the resinwas washed with CH₂Cl₂ and dried, to yield compound 52b.

Compound 53

To the resin was added 410 μL of CH₂Cl₂, and to the mixture was added130 μL of mono-CBZ-piperazine followed by 410 μL of pyridine. Aftergentle agitation of the mixture for 18 hours, the resin was washed withCH₂Cl₂ and dried, to yield compound 53.

Compound 54

To the resin was added 1 mL of 10% TFA in CH₂Cl₂, the mixture wasagitated for 10 min, and the mixture was filtered. The TFA treatment wasrepeated twice, and the combined filtrates were combined andconcentrated to give 3 mg (35%) of compound 54; ¹H NMR (CDCl₃) δ 3.13(m, 4H), 3.48 (m, 8H), 3.80 (m, 4H), 4.50 (m, 1H), 5.18 (s, 4H), 7.37(brd s, 10H); MS (ESI) calculated for C₃₀H₄₀N₅O₈ (M+H): 598. Found: 598.

Compound 40

To a solution of 2.1 eq. of compound 54 and 2.1 eq. of Et₃N in CH₂Cl₂ at0° C. is added a solution of 1 eq. of diethyleneglycol bis-chloroformatein CH₂Cl₂. The mixture is stirred for at room temperature andconcentrated to give crude compound 40 which can be purified by silicagel chromatography.

Example 15 Compound 39b

To a solution of 3.17 g (3.08 mmol) of compound 39 in 35 mL of CH₂Cl2 at0° C. was added 2.6 mL of Et₃N followed by a solution of 3.26 g (18.49mmol) of mono-N-Boc-ethylenediamine (also referred to as tert-butylN-(2-aminoethyl)carbamate, Aldrich Chemical Co.) in 30 mL of CH₂Cl₂. Themixture was stirred at room temperature for 18 hours, cooled to 0°, andacidified with 1 N HCl The mixture was then partitioned between 150 mLof 1 N HCl and three 100 mL portions of CH₂Cl₂. The organic layers werecombined and washed with three portions of saturated sodium bicarbonatesolution, dried (MgSO₄), filtered and concentrated to provide 3.17 g ofyellow solid. Purification by silica gel chromatography (step gradient98/2 to 95/5 to 90/10 CH₂Cl₂/MeOH) provided 2.76 g (80%) of compound 39bas a white solid. ¹H NMR (CDCl₃) δ 1.45 (s, 36H), 3.23 (s, 16H), 3.50(m, 8H), 3.72 (t, 4H), 4.19 (m, 8H), 4.26 (t, 4H), 5.38 (brd s, 4H),5.80 (brd s, 2H), 6.00 (brd s, 2H); mass spectrum (ES) m/z calculatedfor C₄₆H₈₄N₁₀O₂₁ (M+Na): 1 135. Found: 1135.

Compound 39c

A solution of 1 g (9.42 mmol) of diethylene glycol in 20 mL of EtOAc wasadded to a solution of 3.82 g (23.5 mmol) of carbonyldiimidazole in 80mL EtOAc and the resulting mixture was stirred for 2 hours at roomtemperature. The mixture was concentrated to an oily solid, and theproduct was purified by silica gel chromatography (97/2 CH₂Cl₂/MeOH) togive 2.04 g (73%) of the bis-imidazolide of diethylene glycol as a whitesolid: ¹H NMR (CDCl₃) δ 3.87 (t, 4H), 4.58 (t, 4H), 7.08 (s, 2H), 7.41(s, 2H), 8.16 (s, 2H). A solution of 50 mg (0.17 mmol) of thebis-imidazolide of diethylene glycol in 1 mL of CH₂Cl₂ was added to asolution of 54 mg (0.51) mmol) of diethanolamine and 82 μL (80.6 mg,1.02 mmol) of pyridine in 0.5 mL of CH₂Cl₂. The mixture was stirred atroom temperature for four hours, and to the mixture was added a solutionof 248 mg (1.53 mmol) of carbonyldiimidazole in 5 mL of CH₂Cl₂. Themixture was stirred at room temperature for 1.5 hours and concentratedto an oily solid. Purification by silica gel chromatography (98/2CH₂Cl₂/MeOH) provided 103 mg (82%) of the multivalent activatedcarbonate derivative compound 39c, including minor impurities. Theresulting oil crystallized when placed in the freezer: ¹H NMR (CDCl₃) δ3.71 (m, 12H), 4.32 (m, 4H), 4.59 (t, 8H), 7.10 (s, 4H), 7.43 (s, 4H),8.18 (s, 4H); mass spectrum (ES) m/z (relative intensity) 380 (100), 443(18), 512 (26), no parent ion observed.

1-37. (canceled)
 38. A composition comprising valency platformmolecules, wherein each said valency platform molecule comprises atleast 2 carbamate linkages and at least 4 reactive conjugatingfunctional groups; and wherein said valency platform molecules have apolydispersity less than about 1.2.
 39. The composition of claim 38,wherein said valency platform molecules have a polydispersity less thanabout 1.07.
 40. The composition of claim 38, wherein each said valencyplatform molecules comprises at least 4 carbamate linkages and at least8 reactive functional groups.
 41. The composition of claim 38, whereineach said valency platform molecule comprises at least 4 identicalreactive conjugating functional groups.
 42. The composition of claim 38,wherein each said valency platform molecule comprises 2 to 32 carbamatelinkages and 4 to 64 reactive functional groups.
 43. The composition ofclaim 38, wherein comprising said valency platform molecules linked viasaid reactive functional groups to one or more biologically activemolecules.
 44. A composition comprising valency molecules, wherein eachsaid valency molecule comprises at least two branches and at least fourterminal groups; wherein each said valency molecule comprises at least 2carbamate linkages; and wherein said valency molecules have apolydispersity less than about 1.2.
 45. The composition of claim 44,wherein said valency molecules have a polydispersity less than about1.07.
 46. The composition of claim 44, wherein said valency moleculescomprise at least 4 carbamate linkages, at least 4 branches and at least8 terminal groups.